Cystatin C as an antagonist of TGF-β and methods related thereto

ABSTRACT

Disclosed are Cystatin C (CysC) homologues, including CystC homologues that act as antagonists or inhibitors of transforming growth factor-β (TGF-β). Also disclosed are methods to identify CystC homologues that are antagonists or inhibitors of TGF-β and compositions and therapeutic methods using CystC and homologues thereof to regulate the activity of TGF-β, and TGF-β-mediated tumor malignancy and invasion and other TGF-β-mediated fibrotic or proliferative conditions and diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/852,909, filed Sep. 10, 2007, now U.S. Pat. No. 7,749,958, which is acontinuation of U.S. patent application Ser. No. 10/967,093, filed Oct.15, 2004, now U.S. Pat. No. 7,282,477, which claims the benefit ofpriority under 35 U.S.C. §119(e) from U.S. Provisional PatentApplication No. 60/511,794, filed Oct. 15, 2003. The entire disclosureof each application and patent is hereby incorporated by reference forall purposes.

GOVERNMENT RIGHTS

This invention was supported, in part, using funds provided by NIH GrantNo. CA095519, awarded by the National Institutes of Health. Thegovernment has certain rights to this invention.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “2879-104-1_ST25.txt”, having a size in bytes of 20 kb,and created on 10 Sep. 2007. The information contained in thiselectronic file is hereby incorporated by reference in its entiretypursuant to 37 CFR §1.52(e)(5).

FIELD OF THE INVENTION

The present invention generally relates to the use of Cystatin C (CysC)as a regulator of transforming growth factor-β (TGF-β), including as aTGF-β antagonist, and to therapeutic methods associated therewith.

BACKGROUND OF THE INVENTION

The cystatin superfamily of cysteine proteinase inhibitors is comprisedof three major families: Type 1 cystatins, which are cytosolic andinclude stefins A and B; Type 2 cystatins, which are present in mostbodily fluids and include cystatins C, D, E, F, and S; and Type 3cystatins, which are present in plasma and include the kininogens andfetuin (1, 2). Collectively, these molecules inactivate cysteineproteinases and thus regulate (i) bone resorption, neutrophilchemotaxis, and tissue inflammation, (ii) hormone processing and antigenpresentation, and (ii) resistance to bacterial and viral infections(1-3). Cystatin C (CystC) is a ubiquitously expressed, small molecularweight (˜16 kDa) secretory protein that preferentially inactivatescathepsin B, a cysteine proteinase implicated in stimulating cancer cellinvasion and metastasis (4, 5), and in activating latent TGF-β frominactive ECM depots (6, 7). Through its conserved cysteine proteaseinhibitor motif, CystC binds and inactivates cathepsin B by forming areversible, high affinity enzyme-inhibitor complex (8, 9). Independentof its effects on cathepsin B activity, CystC also regulates cellproliferation (10, 11), raising the possibility that CystC targetsproteinase-dependent and -independent pathways.

Mutations in or altered expression of CystC has been linked to thedevelopment and progression of several human pathologies. For instance,a single point mutation in CystC causes Hereditary CystC AmyloidAngiopathy, a lethal autosomal dominant disease that results in massivecerebral hemorrhages in early adulthood (12). Moreover, altered CystCexpression or serum enzyme-inhibitor levels are used as diagnosticmarkers for chronic renal insufficiencies (13), and for cancers of thelung, skin, colon, and myeloid compartment (3, 14-16). Thus, alteredCystC concentrations within cell microenvironments have direconsequences leading to the development and progression of humandiseases.

TGF-β is a multifunctional cytokine that governs cell growth andmotility in part through its regulation of cell microenvironments, andthus plays a prominent role in regulating disease development in humans(17). Critical to regulation of cell microenvironments by TGF-β is itsinduction or repression of cytokines, growth factors, and ECM proteinsby fibroblasts (17).

Transforming growth factor-β (TGF-β) is also a potent suppressor ofmammary epithelial cell (MEC) proliferation, and as such, an inhibitorof mammary tumor formation. However, aberrant genetic and epigeneticevents operant during tumorigenesis typically abrogate the cytostaticfunction of TGF-β, thereby contributing to tumor formation andprogression. For example, malignant MECs typically evolve resistance toTGF-β-mediated growth arrest, thus enhancing their proliferation,invasion, and metastasis when stimulated by TGF-β. Recent findingssuggest that therapeutics designed to antagonize TGF-β signaling mayalleviate breast cancer progression, thereby improving the prognosis andtreatment of breast cancer patients.

Oncogenic epithelial-mesenchymal transitions (EMT) comprise a complexarray of gene expression and repression that elicits tumor metastasis inlocalized carcinomas (Thiery, 2002; Grunert et al., 2003). Theacquisition of metastatic phenotypes by dedifferentiated tumors is themost lethal facet of cancer and the leading cause of cancer-relateddeath (Yoshida et al., 2000; Fidler, 2002). Transforming growth factor-β(TGF-β) normally represses these processes by prohibiting epithelialcell proliferation, and by creating a cell microenvironment thatinhibits epithelial cell motility, invasion, and metastasis (Blobe etal., 2000; Siegel, 2003). Carcinogenesis often subverts the tumorsuppressing function of TGF-β, thereby endowing TGF-β with oncogenicactivities that promote the growth and spread of developing tumors,including the initiation and stabilization of tumor EMT (Thiery, 2002;Grunert et al., 2003; Blobe et al., 2000; Siegel et al., 2003; Wakefieldet al., 2002).

The duality of TGF-β to both suppress and promote cancer development wasobserved originally using transgenic TGF-β1 expression in mousekeratinocytes, which initially suppressed benign skin tumor formationprior to promoting malignant conversion and spindle cell carcinomageneration (Cui et al., 1996). More recently, TGF-β signaling was shownto inhibit the tumorigenicity of normal, premalignant, and malignantbreast epithelial cells, while stimulating that of highly invasive andmetastatic breast cancer cells (Tang et al., 2003). Fundamental gapsexist in the knowledge of how malignant cells overcome the cytostaticactions of TGF-β, and of how TGF-β stimulates the progression ofdeveloping tumors. Indeed, these knowledge gaps have prevented scienceand medicine from developing treatments effective in antagonizing TGF-βoncogenicity in progressing cancers, particularly those of the breast.

TGF-β is widely expressed during development to regulate theinteractions between epithelial and mesenchymal cells, particularlythose in the lung, kidney, and mammary gland. Inappropriate reactivationof EMT during tumorigenesis is now recognized as an important processnecessary for tumor acquisition of invasive and metastatic phenotypes(Thiery, 2002; Grunert et al., 2003). By cooperating with oncogenes andgrowth factors, TGF-β potently induces EMT and serves to stabilize thistransition via autocrine signaling. Moreover, these events appear tounderlie TGF-β oncogenicity and its ability to promote cancerprogression (Miettinen et al., 1994; Oft et al., 1998; Oft et al., 1996;Portella et al., 1998). Molecular dissection of TGF-β signaling systemsnecessary for its induction of EMT has clearly established a role forSmad2/3 in mediating EMT, particularly when coupled with signalsemanating from oncogenic Ras (Piek et al., 1999; Oft et al., 2002; Jandaet al., 2002). However, Smad2/3-independent signaling also has beenimplicated in TGF-β stimulation of EMT. For instance, TGF-β stimulatesEMT in cancers of the breast and other tissues by activatingPI-3-kinase, AKT, RhoA, p160(ROCK), and p38 MAPK (Janda et al., 2002;Bhowmick et al., 2001, Mol. Biol. Cell; Bhowmick et al., 2001, J. Biol.Chem.; Bakin et al., 2000; Yu et al., 2002) In addition, EMT in TGF-βtreated MECs is abrogated by measures that inhibit β1 integrin activity(Bhowmick et al., 2001, J. Biol. Chem.), thus establishing the necessityof β1 integrin expression for EMT stimulated by TGF-β. Finally, byrepressing Id2 and Id3 expression (Kowanetz et al., 2004), inducingSnail and SIP1 expression (Kang et al., 2004), and stimulating NF-κBactivity (Huber et al., 2004), TGF-β regulates transcription factoractivity operant in mediating the transition from epithelial tomesenchymal cell markers. Clearly, EMT and the mechanisms whereby TGF-βparticipates in this process involve a complex cascade of geneexpression and repression, the magnitude of which remains to beelucidated fully.

Accordingly, although TGF-β clearly inhibits the growth and developmentof early stage tumors, an accumulating body of evidence implicates TGF-βsignaling as a stimulus necessary for the metastasis and disseminationof late stage tumors (Blobe et al., 2000; Siegel, 2003). A comprehensiveunderstanding of how TGF-β both suppresses and promotes tumorigenesisremains an unknown and fundamental question that directly impacts ourability to effectively target the TGF-β signaling system duringtreatment of human malignancies. Indeed, deciphering this paradoxremains the most important question concerning the biological andpathological actions of this multifunctional cytokine The ability ofTGF-β to induce cancer growth and metastasis suggests that developingtherapeutics to antagonize and/or circumvent TGF-β signaling may proveeffective in treating metastatic malignancies, perhaps by preventingTGF-β stimulation of EMT.

BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION

FIG. 1A is bar graph showing that CystC expression significantlyinhibited HT1080 cell invasion through Matrigel matrices.

FIG. 1B is a line graph showing that expression of Δ14CystC, which lacksthe cysteine inhibitor motif and thus is unable to inactivate cathepsinB, failed to affect HT1080 cell invasion.

FIG. 2A is a bar graph showing that 3T3-L1 cells that readily invadedthrough Matrigel matrices when stimulated by serum are unaffected byΔ14CystC, but are inhibited significantly by CystC.

FIG. 2B is a bar graph showing that recombinant CystC administrationblocked 3T3-L1 cell invasion stimulated by serum and serum:TGF-β,whereas recombinant Δ14CystC selectively blocked that by TGF-β.

FIG. 3A is a line graph showing that expression of either CystC orΔ14CystC significantly reduced TGF-β-stimulated luciferase activity ascompared to control cells.

FIG. 3B is a bar graph showing that recombinant CystC and Δ14CystC bothsignificantly inhibited luciferase activity stimulated by TGF-β in3T3-L1 cells.

FIG. 4A is a line graph showing CystC that antagonizes TGF-β1 binding toTGF-β receptors.

FIG. 4B is a graph showing that CystC inhibits TGF-β binding to TβR-II.

FIG. 5A is a bar graph showing that recombinant CystC or Δ14CystCtreatment of or their overexpression in NMuMG cells prevented actincytoskeletal reorganization stimulated by TGF-β, as well as antagonizedTGF-β-mediated downregulation of E-cadherin.

FIG. 5B is a bar graph showing that CystC and Δ14CystC expressioninhibited and delineated cathepsin- and TGF-β-dependent invasion inNMuMG cells.

FIG. 6A is a bar graph showing that retroviral-mediated expression ofCystC or Δ14CystC in NRK cells completely prevented morphologicaltransformation stimulated by TGF-β.

FIG. 6B is a bar graph showing that retroviral-mediated expression ofCystC or Δ14CystC in NRK cells completely prevented TGF-β stimulation ofNRK cell invasion through synthetic basement membranes.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to an isolated CystatinC homologue. The homologue comprises an amino acid sequence that is atleast about 50% identical to, or about 60% identical to, or about 70%identical to, or about 80% identical to, or about 90% identical to, anamino acid sequence of a wild-type Cystatin C; and the homologue is lessthan 100% identical to, or less than about 95% identical to, or lessthan about 90% identical to, an amino acid sequence of a wild-typeCystatin C. The Cystatin C homologue inhibits TGF-β biological activity.In one aspect of the invention, the wild-type Cystatin C has an aminoacid sequence represented by SEQ ID NO:2.

In one aspect of this embodiment, the amino acid sequence of thehomologue differs from the amino acid sequence of the wild-type CystatinC protein by a deletion, insertion, substitution or derivatization of atleast one amino acid residue. In another aspect, the amino acid sequenceof the homologue differs from the amino acid sequence of the wild-typeCystatin C protein by a disruption of the wild-type sequence sufficientto reduce or abolish the biological activity of the conserved cysteineproteinase inhibitor motif. For example, the wild-type Cystatin Cprotein can be represented by SEQ ID NO:2 and the conserved cysteineproteinase inhibitor motif can be located between about position 80 andabout position 93 of SEQ ID NO:2. In another aspect, the amino acidsequence of the homologue differs from the amino acid sequence of thewild-type Cystatin C protein represented by SEQ ID NO:2 by a deletion ofamino acid residues from about position 80 to about position 93 withreference to SEQ ID NO:2.

Another embodiment of the present invention relates to an isolatedprotein comprising a fragment of a wild-type Cystatin C protein thatinhibits TGF-β biological activity. In one aspect, the protein inhibitsthe binding of TGF-β to its receptor, including but not limited to,TβRII. In one aspect, the fragment comprises at least about 100 aminoacids, or at least about 75 amino acids, or at least about 55 aminoacids, of the C-terminal portion of the wild-type Cystatin C protein. Inanother aspect, the fragment differs from the wild-type amino acidsequence by a deletion of at least about 10 amino acids, or at leastabout 20 amino acids, or at least about 50 amino acids, from theN-terminus of the wild-type protein.

Another embodiment of the invention relates to a composition, includinga therapeutic composition, comprising any of the above-identifiedhomologues or fragments and a pharmaceutically acceptable carrier.

Yet another embodiment of the present invention relates to a method toinhibit tumor malignancy or invasion, comprising administering to atumor cell Cystatin C or a homologue or synthetic mimetic thereof havingCystatin C biological activity. In one aspect, the biological activitycomprises inhibition of the binding of TGF-β to its receptor. In anotheraspect, the tumor malignancy is cathepsin B-mediated tumor cellmalignancy, and the method comprises contacting a tumor cell withCystatin C or a homologue or synthetic mimetic thereof having Cystatin Cbiological activity effective to inhibit the biological activity ofcathepsin B in the tumor cell or in the microenvironment of the tumorcell. For example, the Cystatin C or homologue or synthetic mimeticthereof can inhibit the biological activity of extracellular cathepsinB. In another aspect, the tumor malignancy is TGF-β-mediated tumormalignancy, and the method comprises contacting a tumor cell withCystatin C or a homologue or synthetic mimetic thereof effective toinhibit the biological activity of TGF-β in the tumor cell or in themicroenvironment of the tumor cell. In yet another aspect, the CystatinC or a homologue or synthetic mimetic thereof inhibits cathepsinB-mediated activation of TGF-β in the cell or tissue or in themicroenvironment of the cell or tissue.

Another embodiment of the present invention relates to a method toinhibit tumor cell malignancy, comprising increasing the expressionand/or biological activity of endogenous Cystatin C in tumor cells.

Yet another embodiment of the present invention relates to a method toinhibit TGF-β biological activity, comprising administering to a cell,tissue or patient Cystatin C or a homologue or synthetic mimeticthereof, or increasing the expression or biological activity ofendogenous Cystatin C, in an amount effective to inhibit biologicalactivity of TGF-β. In one aspect, the method is used to treat a patientwith cancer. In another aspect, the method is used to treat a patientthat has or is predisposed to develop metastatic cancer. In yet anotheraspect, the method is used to treat a patient that has a proliferativeor fibrotic condition or disease mediated at least in part by TGF-βexpression or activity.

Another aspect of the invention relates to a method to increase TGF-βactivity, comprising inhibiting the ability of Cystatin C to regulateTGF-β activity. In one aspect, the method includes contacting a TGF-βreceptor with a Cystatin C homologue that is a competitive inhibitor ofCystatin C. In another aspect, the method includes contacting a TβRIIreceptor with a Cystatin C homologue that is a competitive inhibitor ofCystatin C. In another aspect, the method includes contacting Cystatin Cwith a compound that reduces the ability of Cystatin C to bind to oractivate TβRII. In yet another aspect, the compound is a soluble TβRIIreceptor with a decreased affinity for TGF-β that binds to Cystatin C.In another aspect, the compound binds to Cystatin C and inhibits theability of Cystatin C to bind to TβRII. In another aspect, the compounddoes not inhibit the ability of Cystatin C to inhibit cysteineproteases. Such a compound can include, but is not limited to, anantibody, an antigen binding fragment of an antibody, or a bindingpartner.

Another embodiment of the present invention relates to a method todesign an antagonist or inhibitor of TGF-β activity, comprising: (a)designing or identifying a putative antagonist compound based on thestructure of Cystatin C; (b) synthesizing the compound; and (c)selecting compounds from (b) that inhibit the biological activity ofTGF-β. In one aspect, step (a) comprises performing structure-based drugdesign with a model representing the structure of Cystatin C. In anotheraspect, the step of selecting comprises selecting compounds that inhibitthe binding of TGF-β to its receptor. In one aspect, the step ofselecting comprises selecting compounds that inhibit TGF-β-mediatedtumor cell malignancy or invasion.

Yet another embodiment of the present invention relates to a method toidentify proteins that are inhibitors of TGF-β, comprising: (a)identifying proteins that are structural homologues of Cystatin C; and(b) evaluating proteins from (a) that are capable of regulating thebiological activity of TGF-β. In one aspect, step (a) comprisesidentifying structural homologues or fragments of Cystatin C usingsequence analysis. In another aspect, the method of evaluating comprisesdetecting whether the protein of (a) inhibits the binding of TGF-β toits receptor. In one aspect, the step of selecting comprises selectingcompounds that inhibit TGF-β-mediated tumor cell malignancy or invasion.

Another embodiment of the present invention relates to a method toidentify a regulator of transforming growth factor β (TGF-β),comprising: (a) contacting a cell that expresses a TGF-β receptor andCystatin C with a putative regulatory compound; (b) detecting theexpression of Cystatin C in the cell; and (c) comparing the expressionof

Cystatin C after contact with the compound to the expression of CystatinC before contact with the compound, wherein detection of a change in theexpression of Cystatin C in the cells after contact with the compound ascompared to before contact with the compound indicates that the compoundis a putative regulator of TGF-β and TGF-β signal transduction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to the use of Cystatin C, andbiologically active fragments, homologues and synthetic mimeticsthereof, to regulate the biological activity of TGF-β and TGF-β signaltransduction pathways. The present invention relates to variouscompositions comprising Cystatin C and biologically active fragments,homologues and synthetic mimetics thereof for use in therapeutic,diagnostic and drug screening methods. The present invention relates tothe use of Cystatin C and biologically active fragments, homologues andsynthetic mimetics thereof, to treat or prevent tumor malignancy,including tumor invasion, including tumor malignancy that is mediated bycathepsin-B or by TGF-β. The present invention relates to methods ofregulating Cystatin C expression and/or activity (i.e., up- ordown-regulate) to regulate (as an antagonist or an agonist) thebiological activity of TGF-β or TGF-β signal transduction pathways(e.g., TGF-β receptor signal transduction and the expression, activationand/or biological activity of downstream genes regulated by TGF-β, aswell as regulation of TBF-β receptors). In particular, such methods canbe used to treat or ameliorate at least one symptom of a disease orcondition in which TGF-β plays a role, including cancers and variousproliferative and fibrotic conditions and diseases associated with TGF-βactivity. The present invention also relates to the use of Cystatin Cand biologically active fragments, homologues and synthetic mimeticsthereof as lead compounds for the development of additional antagonists(or agonists) of TGF-β biological activity or of cathepsin-B biologicalactivity. The present invention also relates to the identification ofother members of the Cystatin C supergene family and/or the evaluationof such members for the ability to inhibit TGF-β, followed by the use ofmembers identified as antagonists of TGF-β, including homologues,fragments and synthetic mimetics thereof, in any of the methodsdescribed for Cystatin C herein. The present invention also includes theuse of inhibitors of Cystatin C to regulate the activity of TGF-β or torelease the inhibition of Cystatin C on cysteine proteases (e.g.,cathepsin B). The present invention can include regulation of any of theTGF-β isoforms (TGF-β1, TGF-β2 and/or TGF-β3) by Cystatin C andbiologically active fragments, homologues and synthetic mimeticsthereof. In one embodiment, the biological activity of TGF-β1 or TGF-β3is inhibited. In another embodiment, the biological activity of TGF-β2is inhibited. In yet another embodiment, the biological activity of anyone of TGF-β1, TGF-β2 and/or TGF-β3 is regulated.

Herein the inventor shows that TGF-β stimulates CystC transcript andprotein expression in murine 3T3-L1 fibroblasts. The inventor furthershows that CystC is aberrantly downregulated in human tumors, and thatits overexpression in highly malignant human HT1080 fibrosarcoma cellsinhibits their invasion in a cathepsin B-dependent manner.Interestingly, CystC also inhibits HT1080 cell expression ofTGF-β-responsive genes via a cathepsin B-independent mechanism. Invasionof 3T3-L1 cells proceeds through cathepsin B- and TGF-β-dependentpathways: CystC inhibits both pathways, while a CystC mutant (i.e.,Δ14CystC) unable to inactivate cathepsin B selectively inhibitsTGF-β-dependent invasion, suggesting that CystC targetsproteinase-dependent and -independent signaling pathways. Accordingly,both CystC and Δ14CystC significantly reduce TGF-β-stimulated geneexpression in 3T3-L1 cells by inhibiting the binding of TGF-β to itstype II receptor (TβR-II). Collectively, the findings described hereinestablish a novel CystC-mediated feedback loop that inhibits TGF-βsignaling, doing so by antagonizing TGF-β binding to TβR-II.

More specifically, cell microenvironments play an important role inregulating the physiology and homeostasis of cells, including theirsurvival, proliferation, differentiation, and motility (35). TGF-β is apowerful tumor suppressor that prevents cancer development by inhibitingcell cycle progression, and by creating cell microenvironments thatinhibit uncontrolled cell growth, invasion, and metastasis. Duringcancer progression, the tumor suppressing function of TGF-β isfrequently subverted, thus transforming TGF-β from a suppressor ofcancer formation to a promoter of its growth and metastasis (17, 24).Although mutations in the TGF-β signaling system occur duringcarcinogenesis and contribute to tumor formation by abrogatingTGF-β-mediated cell cycle arrest (17, 24), these aberrances do notexplain the paradoxical function of TGF-β to promote the proliferation,invasion, and metastasis of cancer cells previously liberated from itsgrowth inhibitory actions. Alternatively, recent studies haveestablished fibroblasts as instrumental intermediaries of tumor growthand metastasis stimulated by TGF-β. When activated by TGF-β, fibroblastssynthesize and secrete a variety of cytokines, growth factors, and ECMproteins into tumor:host microenvironments. These secretory proteinsfacilitate tumor:host cross-talk that ultimately promotes the induction,selection, and expansion of neoplastic cells by enhancing their growth,survival, and motility (36). The inventor therefore sought to identifythese TGF-β-regulated fibroblast secretory proteins, and to determinetheir function in normal and cancerous cells.

The inventor now presents CystC as a novel gene target for TGF-β in3T3-L1 fibroblasts. The inventor further shows that CystC expression isprominently downregulated in human cancers (see Example 2), andconversely, that augmenting CystC expression in highly malignantfibrosarcoma cells significantly reduced their invasion throughsynthetic basement membranes (FIG. 1). Most strikingly, these studiesidentified CystC as a novel antagonist of TGF-β signaling (FIGS. 1-3).Indeed, the inventor shows for the first time that CystC inhibits geneexpression (FIGS. 1 and 3) and cellular invasion (FIG. 3) stimulated byTGF-β, doing so by antagonizing TGF-β binding to TβR-II (FIG. 4). Thus,the inventor's study defines a novel cathepsin B-independent functionfor CystC, and has potentially identified a novel CystC-mediatedfeedback loop designed to inhibit TGF-β signaling.

The inactivation of cathepsin B proteinase activity by CystC has been afocal point of numerous investigations aimed at understanding theinvasive properties of cancer cells (19, 37, 38). Although it isgenerally accepted that extracellular cathepsin B mediates cancer cellinvasion, a recent report by Szpaderska and Frankfater (20) has providedevidence to the contrary by demonstrating the importance ofintracellular cathepsin B to cancer cell invasion of through Matrigelmatrices. In contrast, the inventor shows herein that expression ofCystC, and not Δ14CystC, significantly inhibited HT1080 cell invasion.Moreover, the cell permeable cathepsin B inhibitor, CA-074ME, failed toalter HT1080 cell invasion, whereas its cell impermeable counterpart,cathepsin B inhibitor II, fully recapitulated the inhibitory effect ofCystC on HT1080 cell invasion. Thus, HT1080 cell invasion occurspredominantly via the proteinase activities of extracellular cathepsinB, which is subject to inactivation by CystC.

The present inventor's studies also provide the first evidence that3T3-L1 cell invasion proceeds through a bifurcated signaling systemcoupled to the activation of cathepsin B and TGF-β signaling. Moreover,the differential abilities of CystC (i.e., both pathways) and Δ14CystC(i.e., TGF-β pathway) to inhibit 3T3-L1 cell invasion implicates CystCas key regulator not only of cathepsin B-mediated invasion, but alsothat mediated by TGF-β. Although the proteolytic pathways targeted byTGF-β in 3T3-L1 cells remain unknown, without being bound by theory, thepresent inventor suspects involvement of members of the matrixmetalloproteinase family (e.g., MMP-2 and MMP-9) whose expression andactivity are regulated by TGF-β (39). Studies exploiting the anti-TGF-βproperties of Δ14CystC will facilitate the identification and dissectionof this TGF-β-regulated invasive pathway. Indeed, both cathepsin B (19)and TGF-β (40) localize to invading face of highly malignant tumors. Inaddition to its role in cancer cell invasion, cathepsin B also mediateslatent TGF-β activation (6, 7), and as such, would be predicted toenhance the tumor promoting effects of TGF-β. Based on these findings,the inventor proposes that measures designed to deliver CystC todeveloping tumors will reduce their malignancy by inhibiting (i)cathepsin B-mediated invasion, (ii) cathepsin B-mediated TGF-βactivation, and (iii) TGF-β signal transduction.

The present inventor (Example 2) and others (3, 37) have foundtumorigenesis to induce significant downregulation of CystC expression,consistent with a tumor suppressing function for CystC. Recently, micedeficient in CystC expression were generated and found to be viable andexhibit no pronounced abnormalities (41). In addition, no differences inthe latency and growth of subcutaneous and intracerebral tumors weredetected in wild-type and CystC-null mice. Quite surprisingly, lungcolonization by cancer cells was suppressed in CystC-deficient mice ascompared to their wild-type counterparts (41). Although the mechanismmediating this contradictory effect of CystC on cancer cell metastasisis currently unknown, the authors speculate that dysregulated cathepsinB activity suppresses metastasis by catabolizing local cytokines/growthfactors necessary for metastatic cancer cell seeding and growth (41).Based on the present findings, the inventor instead proposes that CystCdeficiency increases tonic TGF-β signaling, resulting in enhanced tumorsuppression and the creation of cell microenvironments that fail toefficiently support the metastatic spread and seeding of cancer cells.Therefore, without being bound by theory, the inventor proposes thatCystC deficiency may potentiate cellular response to TGF-β (i.e., vialoss of CystC-mediated feedback loop).

Finally, the Type 3 cystatin family member, fetuin, has also beencharacterized as a TGF-β antagonist (27-29). Fetuin contains a 19 aminoacid TRH1 domain (TGF-β receptor II homology 1; (29)) that interactsphysically with TGF-β and prevents its binding to TGF-β receptors (29).Although CystC contains C-terminal 21 amino acid sequence havingsimilarity to TRH1, the present inventor was unable to observe a directphysical interaction between TGF-β and CystC. In contrast, the inventorfound that CystC inhibits the binding of TGF-β to TβR-II, both in livecells and in vitro. Interestingly, initial structure-function studies bythe inventor have implicated the C-terminal domain of CystC in mediatingits antagonism of TGF-β signaling. Given the identification of two CystCsuperfamily members capable of inhibiting TGF-β signaling, the inventorproposes that additional CystC superfamily members may similarlyfunction to antagonize the activities of other TGF-β superfamilymembers, as well as the activities of other cytokines/growth factors.Importantly, the present inventor's findings give credence to futurestudies aimed at exploiting the anti-TGF-β properties of CystC (orΔ14CystC) to selectively inhibit the oncogenic activities of TGF-β.Indeed, CystC will form the basis for rationale drug design tofacilitate the development of specific TGF-β receptor antagonistsnecessary to improve the therapeutic response of human malignancies, aswell as a variety of proliferative and fibrotic diseases regulated byTGF-β.

Given these results, the inventor further tested the ability of CystC,as a novel TβR-II antagonist, to block the oncogenic activities ofTGF-β, particularly its ability to stimulate epithelial-mesenchymaltransition (EMT). More specifically, the present inventor tested theoncogenesis-inhibitory properties of CystC by measuring the ability ofCystC to antagonize TGF-β oncogenicity in two established in vitromodels of cancer progression: (i) EMT of normal murine NMuMG mammaryepithelial cells (MEC), and (ii) morphological transformation andanchorage-independent growth of normal rat kidney fibroblasts (NRK). Thepresent inventor demonstrates herein that CystC effectively andcompletely negated TGF-β stimulation of EMT and morphologicaltransformation in mammary and kidney epithelial cells, respectively.Thus, by antagonizing TGF-β signaling and preventing its stimulation ofEMT, CystC is believed to represent a novel TGF-β chemopreventive agenteffective in neutralizing TGF-β oncogenicity and its stimulation oftumor metastasis.

More specifically, as described in the Examples in detail (see Examples8 and 9), CystC and Δ14CystC (a CystC mutant impaired in its ability toinhibit cathepsin protease activity) both inhibited NMuMG cell EMT andinvasion stimulated by TGF-β by preventing actin cytoskeletalrearrangements and E-cadherin downregulation. Moreover, both CystCmolecules completely antagonized TGF-β-mediated morphologicaltransformation and anchorage-independent growth of NRK cells, as well asinhibited their invasion through synthetic basement membranes.Therefore, the present inventor has shown that TGF-β stimulation ofinitiating metastatic events, including decreased cell polarization,reduced cell-cell contact, and elevated cell invasion and migration, areprevented completely by CystC treatment.

Accordingly, the inventor has demonstrated CystC and Δ14CystC bothprevent EMT and morphological transformation stimulated by TGF-β, andthus propose these small molecules as innovative models for thedevelopment of novel TβR-II antagonists designed to combat TGF-βstimulation of tumor progression and EMT. The inventor further proposesthat the chemopreventive effectiveness of CystC will be potentiated byits inhibition of cathepsin B-mediated invasion and metastasis (Turk etal., 2002; Yan et al., 2003; Turk et al., 2000; Roshy et al., 2003), andof cathepsin B-mediated activation of latent TGF-β (Somana et al., 2002;Guo et al., 2002; Gantt et al. 2003), which co-localizes with cathepsinB to the invading face of malignant tumors (Wakefield, 2001; Calkins etal., 1998; Sinha et al., 1995; Sameni et al., 2000). Moreover,CystC-mediated cathepsin B inactivation will reduce the activity of theurokinase plasminogen system, which enhances tumor cell extracellularmatrix degradation, as well as growth factor and latent TGF-β activation(Choong et al., 2003). Cumulatively, the chemopreventive activities ofCystC will antagonize cancer cell response to TGF-β by inhibiting TGF-βbinding, as well as by reducing TGF-β bioavailability within tumormicroenvironments, thereby alleviating TGF-β stimulation of EMT andtumor metastasis in late stage tumors.

In summary, the inventor demonstrated the effectiveness of CystC toinhibit MEC EMT and fibroblast morphological transformation stimulatedby TGF-β. CystC antagonism of TGF-β signaling in MECs occurs independentof its inactivation of cathepsin protease activity, presumably viaCystC:TβR-II complex formation and the prevention of TGF-β binding. Theinventor proposes that CystC or its peptide mimetics ultimately hold thepotential to improve the therapeutic response of human malignanciesregulated by TGF-β, particularly cancers of the breast.

Also encompassed by the invention is the use of inhibitors of CystC toregulate the biological effects of CystC on TGF-β and/or cathepsin B.

It will be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,constructs, or reagents described herein, as such may vary. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the invention that will be limited only by the appended claims.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise. Furthermore, tothe extent that any of the following discussion is general or genetic,it can be applied to any of the proteins, nucleic acids, homologues,fragments and mimetics described herein, as well as to any of themethods described herein.

One embodiment of the present invention relates to Cystatin C proteins,and to homologues and mimetics thereof that are useful in one or moremethods of the present invention, including any methods that takeadvantage of the ability of Cystatin C to regulate TGF-β activity byinterfering with the ability of TGF-β to interact with its receptor. Inparticular, the present invention relates to homologues of Cystatin C,including both protein and synthetic homologues (also called mimetics)that have the biological activity of the wild-type Cystatin C protein,or that at least have the ability to regulate TGF-β activity byinterfering with the ability of TGF-β to interact with its receptor.Compositions and formulations comprising such proteins and homologuesare also encompassed by the invention, as well as methods of using suchproteins and homologues.

As discussed above, Cystatin C (CystC) is a ubiquitously expressed,small molecular weight (˜16 kDa) secretory protein that preferentiallyinactivates cathepsin B, a cysteine proteinase implicated in stimulatingcancer cell invasion and metastasis, and in activating latent TGF-β frominactive ECM depots. Through its conserved cysteine protease inhibitormotif, CystC binds and inactivates cathepsin B by forming a reversible,high affinity enzyme-inhibitor complex. Also, as demonstrated herein,CystC inhibits gene expression and cellular invasion stimulated byTGF-β, doing so by antagonizing TGF-β binding to TβR-II. The nucleotideand amino acid sequences for Cystatin C from a variety of animal speciesare known in the art. For example, the cDNA nucleotide sequence encodinghuman Cystatin C is found in NCBI Database GI No. 19882253 and isrepresented herein by SEQ ID NO:1. SEQ ID NO:1 encodes the humanCystatin C amino acid sequence represented herein by SEQ ID NO:2. ThecDNA nucleotide sequence encoding murine Cystatin C is found in NCBIDatabase GI No. 31981821 and is represented herein by SEQ ID NO:3. SEQID NO:3 encodes the murine Cystatin C amino acid sequence representedherein by SEQ ID NO:4. The cDNA nucleotide sequence encoding ratCystatin C is found in NCBI Database GI No. 34858909 and is representedherein by SEQ ID NO:5. SEQ ID NO:5 encodes the rat Cystatin C amino acidsequence represented herein by SEQ ID NO:6. The cDNA nucleotide sequenceencoding bovine Cystatin C is found in NCBI Database GI No. 27806674 andis represented herein by SEQ ID NO:7. SEQ ID NO:7 encodes the bovineCystatin C amino acid sequence represented herein by SEQ ID NO:8. ThecDNA nucleotide sequence encoding rhesus monkey Cystatin C is found inNCBI Database GI No. 2281118 and is represented herein by SEQ ID NO:9.SEQ ID NO:9 encodes the rhesus monkey Cystatin C amino acid sequencerepresented herein by SEQ ID NO:10. There is a high degree of homologyamong Cystatin C proteins from these various animal species. Forexample, the murine, rat, bovine and rhesus monkey amino acid sequencesare about 67%, 68%, 68%, and 97% identical, respectively, to the humanCystatin C amino acid sequence over the full length of such sequences.Human Cystatin C has been crystallized and the structure determined, andthe atomic coordinates for the tertiary structure of Cystatin C is foundin the Protein Database Accession No. 1G96 (described in and depositedby Janowski et al., Nat. Struct. Biol. 8(4):316-320, 2001), incorporatedherein by reference in its entirety.

An isolated protein, according to the present invention, is a proteinthat has been removed from its natural milieu (i.e., that has beensubject to human manipulation) and can include purified proteins,partially purified proteins, recombinantly produced proteins, andsynthetically produced proteins, for example. As such, “isolated” doesnot reflect the extent to which the protein has been purified.Preferably, an isolated protein of the present invention is producedrecombinantly. Reference to a particular protein from a specificorganism, such as a “human Cystatin C protein”, by way of example,refers to a Cystatin C protein (including a homologue of a naturallyoccurring Cystatin C protein) from a human or a Cystatin C protein thathas been otherwise produced from the knowledge of the structure (e.g.,sequence) of a naturally occurring Cystatin C protein from a human. Inother words, a human Cystatin C protein includes any Cystatin C proteinthat has the structure and function of a naturally occurring Cystatin Cprotein from a human or that has a structure and function that issufficiently similar to a human Cystatin C protein such that theCystatin C protein is a biologically active (i.e., has biologicalactivity) homologue of a naturally occurring Cystatin C protein from ahuman. As such, a human Cystatin C protein can include purified,partially purified, recombinant, mutated/modified and syntheticproteins.

In general, the biological activity or biological action of a proteinrefers to any function(s) exhibited or performed by the protein that isascribed to the naturally occurring form of the protein as measured orobserved in vivo (i.e., in the natural physiological environment of theprotein) or in vitro (i.e., under laboratory conditions). Modificationsof a protein, such as in a homologue or mimetic (discussed below), mayresult in proteins having the same biological activity as the naturallyoccurring protein, or in proteins having decreased or increasedbiological activity as compared to the naturally occurring protein.Modifications which result in a decrease in protein expression or adecrease in the activity of the protein, can be referred to asinactivation (complete or partial), down-regulation, or decreased actionof a protein. Similarly, modifications which result in an increase inprotein expression or an increase in the activity of the protein, can bereferred to as amplification, overproduction, activation, enhancement,up-regulation or increased action of a protein.

According to the present invention, Cystatin C biological activity caninclude one or more (or all) of the following biological activities ofwild-type Cystatin C: inhibition of a cysteine protease, andparticularly cathepsin B (e.g., by binding to and inactivating cathepsinB by forming a reversible, high affinity enzyme-inhibitor complex);regulation of cell proliferation; binding to a TGF-β receptor (e.g.,binding to TβRII and thereby preventing TGF-β activation of TβRII); andinhibition of TGF-β stimulation of initiating metastatic events,including decreased cell polarization, reduced cell-cell contact, andelevated cell invasion and migration. According to the presentinvention, CystC biological activity can include the regulation of theactivity of any isoform of TGF-β, including TGF-β1, TGF-β2 and TGF-β3.Similarly, CystC biological activity can include antagonizing thebinding if TGF-β to any receptor of a TGF-β protein, including, but notlimited to, TβR-I, TβR-II, and TβR-III. The present inventor believes,without being bound by theory, that CystC can antagonize TGF-β bindingby binding more specifically to TβRII, and particularly to theextracellular ligand binding domain of TβRII, but the effect can includeinhibition of any of the three receptors mentioned above.

Methods of detecting and measuring protein expression and biologicalactivity include, but are not limited to, measurement of transcriptionof a protein, measurement of translation of a protein, measurement ofposttranslational modification of a protein, measurement of the abilityof the protein to bind to another protein(s); measurement of the abilityof the protein to induce or participate in a particular biologicaleffect (e.g., for Cystatin C, inhibition of the activity cathepsin B,inhibition of the binding of TGF-β to TβRII, regulation of cellularproliferation, inhibition of TGF-β and/or cathepsin B-dependent tumorcell malignancy and invasion). It is noted that an isolated protein ofthe present invention (including a homologue) is not necessarilyrequired to have the biological activity of the wild-type protein. Forexample, a protein can be a truncated, mutated or inactive protein, forexample. Such proteins are useful in screening assays or diagnosticassays, for example, or for other purposes such as antibody production.In a preferred embodiment, the isolated proteins of the presentinvention have a biological activity that is similar to that of thewild-type protein (although not necessarily equivalent, as discussedbelow).

Methods to measure protein expression levels generally include, but arenot limited to: Western blot, immunoblot, enzyme-linked immunosorbantassay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surfaceplasmon resonance, chemiluminescence, fluorescent polarization,phosphorescence, immunohistochemical analysis, matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) mass spectrometry,microcytometry, microarray, microscopy, fluorescence activated cellsorting (FACS), and flow cytometry, as well as assays based on aproperty of the protein including but not limited to enzymatic activityor interaction with other protein partners. Binding assays are also wellknown in the art. For example, a BIAcore machine can be used todetermine the binding constant of a complex between two proteins. Thedissociation constant for the complex can be determined by monitoringchanges in the refractive index with respect to time as buffer is passedover the chip (O'Shannessy et al. Anal. Biochem. 212:457-468 (1993);Schuster et al., Nature 365:343-347 (1993)). Other suitable assays formeasuring the binding of one protein to another include, for example,immunoassays such as enzyme linked immunoabsorbent assays (ELISA) andradioimmunoassays (RIA); or determination of binding by monitoring thechange in the spectroscopic or optical properties of the proteinsthrough fluorescence, UV absorption, circular dichroism, or nuclearmagnetic resonance (NMR).

Methods suitable for use in measuring the biological activity ofCystatin C or homologues and mimetics thereof of the invention include,but are not limited to: binding assays (described above and in theExamples) for CystC binding to cathepsin C, to another cysteineprotease, or to TβRII; assays for measuring the effect of CystC oncysteine protease activity (e.g., enzyme assays); assays for measuringthe effect of CystC on gene expression (e.g., Northern blot, Westernblot, microarray studies); assays for measuring the effect of CystC onTGF-β activity (e.g., reporter gene assays, proliferation assays,receptor binding assays, immunofluorescence assays); assays formeasuring the effect of CystC on tumor cell malignancy and invasion(e.g., tumor cell invasion assays, soft agar assays, morphologicalanalyses, immunofluorescence assays). Other assays for CystC biologicalactivity based on the disclosure provided herein will be apparent tothose of skill in the art. A variety of different assays for determiningand measuring the activity of CystC and homologues thereof is describedin the Examples.

The present invention includes homologues of various proteins describedherein (e.g., Cystatin C protein). As used herein, the term “homologue”is used to refer to a protein or peptide which differs from a naturallyoccurring protein or peptide (i.e., the “prototype” or “wild-type”protein) by one or more minor modifications or mutations to thenaturally occurring protein or peptide, but which maintains the overallbasic protein and side chain structure of the naturally occurring form(i.e., such that the homologue is identifiable as being related to thewild-type protein). Such changes include, but are not limited to:changes in one or a few amino acid side chains; changes one or a fewamino acids, including deletions (e.g., a truncated version of theprotein or peptide) insertions and/or substitutions; changes instereochemistry of one or a few atoms; and/or minor derivatizations,including but not limited to: methylation, farnesylation, geranylgeranylation, glycosylation, carboxymethylation, phosphorylation,acetylation, myristoylation, prenylation, palmitation, and/or amidation.A homologue can have either enhanced, decreased, or substantiallysimilar properties as compared to the naturally occurring protein orpeptide. Preferred homologues of a Cystatin C protein are described indetail below. It is noted that homologues can include syntheticallyproduced homologues (synthetic peptides or proteins), naturallyoccurring allelic variants of a given protein, or homologous sequencesfrom organisms other than the organism from which the reference sequencewas derived.

Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine and leucine;aspartic acid, glutamic acid, asparagine, and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine.Substitutions may also be made on the basis of conserved hydrophobicityor hydrophilicity (Kyte and Doolittle, J. Mol. Biol. (1982) 157:105-132), or on the basis of the ability to assume similar polypeptidesecondary structure (Chou and Fasman, Adv. Enzymol. (1978) 47: 45-148,1978).

Homologues can be the result of natural allelic variation or naturalmutation. A naturally occurring allelic variant of a nucleic acidencoding a protein is a gene that occurs at essentially the same locus(or loci) in the genome as the gene which encodes such protein, butwhich, due to natural variations caused by, for example, mutation orrecombination, has a similar but not identical sequence. Allelicvariants typically encode proteins having similar activity to that ofthe protein encoded by the gene to which they are being compared. Oneclass of allelic variants can encode the same protein but have differentnucleic acid sequences due to the degeneracy of the genetic code.Allelic variants can also comprise alterations in the 5′ or 3′untranslated regions of the gene (e.g., in regulatory control regions).Allelic variants are well known to those skilled in the art.

Homologues can be produced using techniques known in the art for theproduction of proteins including, but not limited to, directmodifications to the isolated, naturally occurring protein, directprotein synthesis, or modifications to the nucleic acid sequenceencoding the protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis.

Modifications or mutations in protein homologues, as compared to thewild-type protein, either increase, decrease, or do not substantiallychange, the basic biological activity of the homologue as compared tothe naturally occurring (wild-type) protein. With regard to Cystatin C,the present invention includes homologues that maintain the basicbiological activities of the wild-type protein, as well as homologuesthat maintain only some of the biological activities of the wild-typeprotein (e.g., the ability to regulate TGF-β activity, but not cathepsinB activity). An example of such a homologue is described in the Examples(i.e., the Δ14CystC modified protein). Biological activities of CystatinC have been described in detail elsewhere herein. Modifications of aprotein, such as in a homologue or mimetic (discussed below), may resultin proteins having the same biological activity as the naturallyoccurring protein, or in proteins having decreased or increasedbiological activity as compared to the naturally occurring protein.Modifications which result in a decrease in protein expression or adecrease in the activity of the protein, can be referred to asinactivation (complete or partial), down-regulation, or decreased action(or activity) of a protein. Similarly, modifications which result in anincrease in protein expression or an increase in the activity of theprotein, can be referred to as amplification, overproduction,activation, enhancement, up-regulation or increased action (or activity)of a protein. It is noted that general reference to a homologue havingthe biological activity of the wild-type protein does not necessarilymean that the homologue has identical biological activity as thewild-type protein, particularly with regard to the level of biologicalactivity. Rather, a homologue can perform the same general biologicalactivity as the wild-type protein, but at a reduced or increased levelof activity as compared to the wild-type protein. A functional domain ofa Cystatin C protein is a domain (i.e., a domain can be a portion of aprotein) that is capable of performing a biological function (i.e., hasbiological activity, such as the ability to bind to a TGF-β receptor,including TβRII).

In one preferred embodiment of the present invention a CystC homologuehas an amino acid sequence that differs from the amino acid sequence ofthe wild-type Cystatin C protein by a disruption (e.g., deletion,substitution, insertion) of the wild-type sequence sufficient to reduceor abolish the biological activity of the conserved cysteine proteinaseinhibitor motif. According to the present invention, the conservedcysteine proteinase inhibitor motif of human CystC, for example, islocated between about position 80 and about position 93 of CystC. In oneaspect, the amino acid sequence of the homologue differs from the aminoacid sequence of the wild-type Cystatin C protein by a deletion of aminoacid residues from about position 50 to about position 120 withreference to the wild-type amino acid sequence of human CystC. Inanother aspect, the amino acid sequence of the homologue differs fromthe amino acid sequence of the wild-type Cystatin C protein by adeletion of amino acid residues from about position 60 to about position110, or from about position 70 to about position 100, or from aboutposition 80 to about position 93, with reference to the wild-type aminoacid sequence of human CystC. Also encompassed are any length deletionsbetween about position 50 and 120 that include the about position 80 toabout position 93 deletion, with reference to the human Cyst C aminoacid sequence (SEQ ID NO:2). A human CystC having a deletion fromposition 80 to position 93 is described in the Examples and referred toherein as Δ14CystC. Δ14CystC does not bind to cathepsin B and thereforedoes not have the cysteine protease inhibitory activity of wild-typeCystC. However, Δ14CystC retains the ability to regulate the activity ofTGF-β comparable to the wild-type protein. Such activity (or lackthereof) can be used to evaluate other deletion mutants of CystC asdescribed above. The corresponding positions in other CystC proteinsfrom other species can readily be determined by one of skill in the art,for example, by alignment of the human sequence with the other sequence(described elsewhere herein). With regard to the TGF-β-regulatingactivity of CystC, the present inventor has determined that thisactivity is correlated with residues in the C-terminal region of CystC.Therefore, a CystC homologue of the invention (including a biologicallyactive fragment thereof) preferably retains the C-terminal portion ofthe wild-type protein, and most preferably, at least the last 50C-terminal amino acids, or at least the last 45 C-terminal amino acids,or at least the last 40 C-terminal amino acids, or at least the last 35C-terminal amino acids, or at least the last 30 C-terminal amino acids,or at least the last 25 C-terminal amino acids, and preferably at leastthe last 21 C-terminal amino acids of the wild-type sequence. Theretention of the TGF-β-regulating activity of a CystC homologue canreadily be evaluated, for example, by determining whether the homologuecan bind to a TGF-β receptor such as TβRII (described herein).

In one embodiment of the invention, a homologue of CystC useful in themethods of the invention includes a fragment of the full-length Cyst C.In one embodiment, such a fragment consists essentially of or consistsof a fragment of a wild-type Cystatin C protein that is capable ofinhibiting TGF-β biological activity. In one embodiment, the fragmentdiffers from the wild-type amino acid sequence by a deletion of at leastabout 10 amino acids from the N-terminus of the wild-type protein, or adeletion of at least about 15 amino acids from the N-terminus, or adeletion of at least about 20 amino acids from the N-terminus, or adeletion of at least about 25 amino acids from the N-terminus, or adeletion of at least about 30 amino acids from the N-terminus, or adeletion of at least about 35 amino acids from the N-terminus, or adeletion of at least about 40 amino acids from the N-terminus, or adeletion of at least about 45 amino acids from the N-terminus, or adeletion of at least about 50 amino acids from the N-terminus, or adeletion of at least about 55 amino acids from the N-terminus, or adeletion of at least about 60 amino acids from the N-terminus, or adeletion of at least about 65 amino acids from the N-terminus, or adeletion of at least about 70 amino acids from the N-terminus, or adeletion of at least about 80 amino acids from the N-terminus, or adeletion of at least about 85 amino acids from the N-terminus, or adeletion of at least about 90 amino acids from the N-terminus, or adeletion of at least about 95 amino acids from the N-terminus, or adeletion of at least about 100 amino acids from the N-terminus of thewild-type CystC protein.

In one aspect of the invention, a homologue of CystC comprises, consistsessentially of, or consists of, an amino acid sequence that is at leastabout 50% identical, and more preferably at least about 55% identical,and more preferably at least about 60% identical, and more preferably atleast about 65% identical, and more preferably at least about 70%identical, and more preferably at least about 75% identical, and morepreferably at least about 80% identical, and even more preferably atleast about 85% identical, and even more preferably at least about 90%identical and even more preferably at least about 95% identical, andeven more preferably at least about 96% identical, and even morepreferably at least about 97% identical, and even more preferably atleast about 98% identical, and even more preferably at least about 99%identical (or any percentage between 60% and 99%, in whole singlepercentage increments) to the natural reference amino acid sequence(e.g., the wild-type CystC protein, such as that represented by SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10) over alength of the natural sequence that is at least the same as the lengthof the homologue. A homologue includes a fragment of a natural(full-length or wild-type sequence), including biologically active,partially biologically active (e.g., binds to a ligand or receptor, butmay not have further biological activity), biologically inactive, andsoluble forms of the natural protein (e.g., if the natural protein is amembrane or insoluble protein).

In one embodiment, a CystC homologue of the present invention comprises,consists essentially of, or consists of an amino acid sequence that isless than 100% identical to the wild-type sequence for CystC, or lessthan about 99% identical, or less than 98% identical, or less than 97%identical, or less than 96% identical, or less than 95% identical, orless than 94% identical, or less than 93% identical, or less than 92%identical, or less than 91% identical, or less than 90% identical to thewild-type CystC sequence (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8 or SEQ ID NO:10), and so on, in increments of wholeintegers. The isolated CystC homologue of the present inventionpreferably has at least one biological activity of a naturally occurringor wild-type CystC protein.

As used herein, unless otherwise specified, reference to a percent (%)identity refers to an evaluation of homology which is performed using aBLAST homology search. BLAST homology searches can be performed usingthe BLAST database and software, which offers search programs including:(1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acidsearches and blastn for nucleic acid searches with standard defaultparameters, wherein the query sequence is filtered for low complexityregions by default (described in Altschul, S. F., Madden, T. L.,Schääeffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.(1997) “Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs.” Nucleic Acids Res. 25:3389-3402, incorporated hereinby reference in its entirety); (2) a BLAST 2 alignment (using theparameters described below); (3) and/or PSI-BLAST with the standarddefault parameters (Position-Specific Iterated BLAST. It is noted thatdue to some differences in the standard parameters between BLAST 2.0Basic BLAST and BLAST 2, two specific sequences might be recognized ashaving significant homology using the BLAST 2 program, whereas a searchperformed in BLAST 2.0 Basic BLAST using one of the sequences as thequery sequence may not identify the second sequence in the top matches.

Two specific sequences can be aligned to one another using BLAST 2sequence as described in Tatusova and Madden, (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250, incorporated herein by reference inits entirety. BLAST 2 sequence alignment is performed in blastp orblastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search(BLAST 2.0) between the two sequences allowing for the introduction ofgaps (deletions and insertions) in the resulting alignment. For purposesof clarity herein, a BLAST 2 sequence alignment is performed using thestandard default parameters as follows.

For blastn, using 0 BLOSUM62 matrix:

-   -   Reward for match=1    -   Penalty for mismatch=−2    -   Open gap (5) and extension gap (2) penalties    -   gap x_dropoff (50) expect (10) word size (11) filter (on)        For blastp, using 0 BLOSUM62 matrix:    -   Open gap (11) and extension gap (1) penalties    -   gap x_dropoff (50) expect (10) word size (3) filter (on).

In addition, PSI-BLAST provides an automated, easy-to-use version of a“profile” search, which is a sensitive way to look for sequencehomologues. The program first performs a gapped BLAST database search.The PSI-BLAST program uses the information from any significantalignments returned to construct a position-specific score matrix, whichreplaces the query sequence for the next round of database searching.Therefore, it is to be understood that percent identity can bedetermined by using any one of these programs, although for the directcomparison of two sequences, BLAST 2 is preferred.

According to the present invention, the term “contiguous” or“consecutive”, with regard to nucleic acid or amino acid sequencesdescribed herein, means to be connected in an unbroken sequence. Forexample, for a first sequence to comprise 30 contiguous (or consecutive)amino acids of a second sequence, means that the first sequence includesan unbroken sequence of 30 amino acid residues that is 100% identical toan unbroken sequence of 30 amino acid residues in the second sequence.Similarly, for a first sequence to have “100% identity” with a secondsequence means that the first sequence exactly matches the secondsequence with no gaps between nucleotides or amino acids.

In another embodiment, an isolated protein of the present invention,including an isolated homologue, includes a protein having an amino acidsequence that is sufficiently similar to a naturally occurring proteinamino acid sequence that a nucleic acid sequence encoding the homologueis capable of hybridizing under moderate, high, or very high stringencyconditions (described below) to (i.e., with) a nucleic acid moleculeencoding the naturally occurring protein (i.e., to the complement of thenucleic acid strand encoding the naturally occurring protein amino acidsequence). A “complement” of nucleic acid sequence encoding a protein ofthe present invention refers to the nucleic acid sequence of the nucleicacid strand that is complementary to the strand which encodes theprotein. Methods to deduce a complementary sequence are well known tothose skilled in the art. It should be noted that since amino acidsequencing and nucleic acid sequencing technologies are not entirelyerror-free, the sequences presented herein, at best, represent apparentsequences of PUFA PKS domains and proteins of the present invention.

As used herein, hybridization conditions refer to standard hybridizationconditions under which nucleic acid molecules are used to identifysimilar nucleic acid molecules. Such standard conditions are disclosed,for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., isincorporated by reference herein in its entirety (see specifically,pages 9.31-9.62). In addition, formulae to calculate the appropriatehybridization and wash conditions to achieve hybridization permittingvarying degrees of mismatch of nucleotides are disclosed, for example,in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al.,ibid., is incorporated by reference herein in its entirety.

More particularly, low stringency hybridization and washing conditions,as referred to herein, refer to conditions which permit isolation ofnucleic acid molecules having at least about 50% nucleic acid sequenceidentity with the nucleic acid molecule being used to probe in thehybridization reaction (i.e., conditions permitting about 50% or lessmismatch of nucleotides). Moderate stringency hybridization and washingconditions, as referred to herein, refer to conditions which permitisolation of nucleic acid molecules having at least about 70% nucleicacid sequence identity with the nucleic acid molecule being used toprobe in the hybridization reaction (i.e., conditions permitting about30% or less mismatch of nucleotides). High stringency hybridization andwashing conditions, as referred to herein, refer to conditions whichpermit isolation of nucleic acid molecules having at least about 80%nucleic acid sequence identity with the nucleic acid molecule being usedto probe in the hybridization reaction (i.e., conditions permittingabout 20% or less mismatch of nucleotides). Very high stringencyhybridization and washing conditions, as referred to herein, refer toconditions which permit isolation of nucleic acid molecules having atleast about 90% nucleic acid sequence identity with the nucleic acidmolecule being used to probe in the hybridization reaction (i.e.,conditions permitting about 10% or less mismatch of nucleotides). Asdiscussed above, one of skill in the art can use the formulae inMeinkoth et al., ibid. to calculate the appropriate hybridization andwash conditions to achieve these particular levels of nucleotidemismatch. Such conditions will vary, depending on whether DNA:RNA orDNA:DNA hybrids are being formed. Calculated melting temperatures forDNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particularembodiments, stringent hybridization conditions for DNA:DNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 20° C. and about 35° C. (lower stringency),more preferably, between about 28° C. and about 40° C. (more stringent),and even more preferably, between about 35° C. and about 45° C. (evenmore stringent), with appropriate wash conditions. In particularembodiments, stringent hybridization conditions for DNA:RNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 30° C. and about 45° C., more preferably,between about 38° C. and about 50° C., and even more preferably, betweenabout 45° C. and about 55° C., with similarly stringent wash conditions.These values are based on calculations of a melting temperature formolecules larger than about 100 nucleotides, 0% formamide and a G+Ccontent of about 40%. Alternatively, T_(m) can be calculated empiricallyas set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general,the wash conditions should be as stringent as possible, and should beappropriate for the chosen hybridization conditions. For example,hybridization conditions can include a combination of salt andtemperature conditions that are approximately 20-25° C. below thecalculated T_(m) of a particular hybrid, and wash conditions typicallyinclude a combination of salt and temperature conditions that areapproximately 12-20° C. below the calculated T_(m) of the particularhybrid. One example of hybridization conditions suitable for use withDNA:DNA hybrids includes a 2-24 hour hybridization in 6×SSC (50%formamide) at about 42° C., followed by washing steps that include oneor more washes at room temperature in about 2×SSC, followed byadditional washes at higher temperatures and lower ionic strength (e.g.,at least one wash as about 37° C. in about 0.1×-0.5×SSC, followed by atleast one wash at about 68° C. in about 0.1×-0.5×SSC).

Proteins of the present invention also include expression products ofgene fusions (for example, used to overexpress soluble, active forms ofthe recombinant protein), of mutagenized genes (such as genes havingcodon modifications to enhance gene transcription and translation), andof truncated genes (such as genes having membrane binding domainsremoved to generate soluble forms of the membrane protein, or geneshaving signal sequences removed which are poorly tolerated in aparticular recombinant host).

The present invention also includes a fusion protein or a chimericprotein that includes a desired protein-containing domain (e.g., CystCor a homologue or fragment thereof) attached to one or more fusionsegments or additional proteins or peptides. Suitable fusion segmentsfor use with the present invention include, but are not limited to,segments that can: enhance a protein's stability; provide otherdesirable biological activity; and/or assist with the purification of aprotein (e.g., by affinity chromatography), or provide another proteinfunction (e.g., as in a chimeric protein). A suitable fusion segment canbe a domain of any size that has the desired function (e.g., impartsincreased stability, solubility, action or biological activity;simplifies purification of a protein; or provides the additional proteinfunction). Fusion segments can be joined to amino and/or carboxyltermini of the domain of the desired protein and can be susceptible tocleavage in order to enable straight-forward recovery of the protein. Inone embodiment a suitable fusion segment or protein with which achimeric or fusion protein can be produced is an antibody fragment andparticularly, the Fc portion of an immunoglobulin protein. Any fusion orchimera partner that enhances the stability or half-life of CystC invivo, for example, is contemplated for use in the present invention.

In one embodiment of the present invention, any of the above-describedamino acid sequences, as well as homologues of such sequences, can beproduced with from at least one, and up to about 20, additionalheterologous amino acids flanking each of the C- and/or N-terminal endof the given amino acid sequence. The resulting protein or polypeptidecan be referred to as “consisting essentially of” a given amino acidsequence. According to the present invention, the heterologous aminoacids are a sequence of amino acids that are not naturally found (i.e.,not found in nature, in vivo) flanking the given amino acid sequence orwhich would not be encoded by the nucleotides that flank the naturallyoccurring nucleic acid sequence encoding the given amino acid sequenceas it occurs in the gene, if such nucleotides in the naturally occurringsequence were translated using standard codon usage for the organismfrom which the given amino acid sequence is derived. Similarly, thephrase “consisting essentially of”, when used with reference to anucleic acid sequence herein, refers to a nucleic acid sequence encodinga given amino acid sequence that can be flanked by from at least one,and up to as many as about 60, additional heterologous nucleotides ateach of the 5′ and/or the 3′ end of the nucleic acid sequence encodingthe given amino acid sequence. The heterologous nucleotides are notnaturally found (i.e., not found in nature, in vivo) flanking thenucleic acid sequence encoding the given amino acid sequence as itoccurs in the natural gene.

The minimum size of a protein and/or a homologue or fragment thereof ofthe present invention is, in one aspect, a size sufficient to have therequisite biological activity, or sufficient to serve as an antigen forthe generation of an antibody or as a target in an in vitro assay. Inone embodiment, a protein of the present invention is at least about 8amino acids in length (e.g., suitable for an antibody epitope or as adetectable peptide in an assay), or at least about 25 amino acids inlength, or at least about 30 amino acids in length, or at least about 35amino acids in length, or at least about 40 amino acids in length, or atleast about 50 amino acids in length, or at least about 60 amino acidsin length, or at least about 65 amino acids in length, or at least about70 amino acids in length, or at least about 75 amino acids in length, orat least about 80 amino acids in length, or at least about 85 aminoacids in length, or at least about 90 amino acids in length, or at leastabout 95 amino acids in length, or at least about 100 amino acids inlength, or at least about 105 amino acids in length, or at least about110 amino acids in length, or at least about 115 amino acids in length,or at least about 120 amino acids in length, or at least about 125 aminoacids in length, or at least about 130 amino acids in length, or atleast about 140 amino acids in length, or at least about 145 amino acidsin length, and so on, in any length between 8 amino acids and up to thefull length of a protein of the invention or longer, in whole integers(e.g., 8, 9, 10, . . . 25, 26, . . . 102, 103, . . . ). There is nolimit, other than a practical limit, on the maximum size of such aprotein in that the protein can include a portion of a protein, afunctional domain, or a biologically active or useful fragment thereof,or a full-length protein, plus additional sequence (e.g., a fusionprotein sequence), if desired.

Another embodiment of the present invention relates to a compositioncomprising at least about 500 ng, and preferably at least about 1 μg,and more preferably at least about 5 μg, and more preferably at leastabout 10 μg, and more preferably at least about 25 μg, and morepreferably at least about 50 μg, and more preferably at least about 75μg, and more preferably at least about 100 μg, and more preferably atleast about 250 μg, and more preferably at least about 500 μg, and morepreferably at least about 750 μg, and more preferably at least about 1mg, and more preferably at least about 5 mg, of an isolated CystCprotein or homologue or mimetic thereof comprising any of the CystCproteins or homologues thereof discussed herein. Such a composition ofthe present invention can include any carrier with which the protein isassociated by virtue of the protein preparation method, a proteinpurification method, or a preparation of the protein for use in an invitro, ex vivo, or in vivo method according to the present invention.For example, such a carrier can include any suitable excipient, bufferand/or delivery vehicle, such as a pharmaceutically acceptable carrier(discussed below), which is suitable for combining with the CystCprotein of the present invention so that the protein can be used invitro, ex vivo or in vivo according to the present invention.

Homologues of a protein described herein such as CystC homologues,including peptide and non-peptide agonists and antagonists of CystC, canbe products of drug design or selection and can be produced usingvarious methods known in the art. Such homologues can more particularlybe referred to as mimetics. A mimetic refers to any peptide ornon-peptide compound that is able to mimic the biological action of anaturally occurring peptide, often because the mimetic has a basicstructure that mimics the basic structure of the naturally occurringpeptide and/or has the salient biological properties of the naturallyoccurring peptide. Mimetics can include, but are not limited to:peptides that have substantial modifications from the prototype such asno side chain similarity with the naturally occurring peptide (suchmodifications, for example, may decrease its susceptibility todegradation); anti-idiotypic and/or catalytic antibodies, or fragmentsthereof; non-proteinaceous portions of an isolated protein (e.g.,carbohydrate structures); or synthetic or natural organic molecules,including nucleic acids and drugs identified through combinatorialchemistry, for example. Such mimetics can be designed, selected and/orotherwise identified using a variety of methods known in the art.Various methods of drug design, useful to design or select mimetics orother therapeutic compounds useful in the present invention aredisclosed in Maulik et al., 1997, Molecular Biotechnology: TherapeuticApplications and Strategies, Wiley-Liss, Inc., which is incorporatedherein by reference in its entirety.

An agonist can be any compound which is capable of mimicking,duplicating or approximating the biological activity of a naturallyoccurring or specified protein, for example, by associating with (e.g.,binding to) or activating a protein (e.g., a receptor) to which thenatural protein binds, so that activity that would be produced with thenatural protein is stimulated, induced, increased, or enhanced. Forexample, an agonist can include, but is not limited to, a protein,compound, or an antibody that selectively binds to and activates orincreases the activation of a receptor bound by the natural protein,other homologues of the natural protein, and any suitable product ofdrug design that is characterized by its ability to agonize (e.g.,stimulate, induce, increase, enhance) the biological activity of anaturally occurring protein.

An antagonist refers to any compound or agent which is capable of actingin a manner that is antagonistic (e.g., against, a reversal of, contraryto) to the action of the natural agonist, for example by interactingwith another protein or molecule in a manner that the biologicalactivity of the naturally occurring protein or agonist is decreased(e.g., reduced, inhibited, blocked). Such a compound can include, but isnot limited to, an antibody that selectively binds to and blocks accessto a protein by its natural ligand, or reduces or inhibits the activityof a protein, a product of drug design that blocks the protein orreduces the biological activity of the protein, an anti-sense nucleicacid molecule that binds to a nucleic acid molecule encoding the proteinand prevents expression of the protein, a ribozyme that binds to the RNAand prevents expression of the protein, and a soluble protein, whichcompetes with a natural receptor or ligand.

A mimetic, including agonists and antagonists, can be produced usingvarious methods known in the art. A mimetic can be obtained, forexample, from molecular diversity strategies (a combination of relatedstrategies allowing the rapid construction of large, chemically diversemolecule libraries), libraries of natural or synthetic compounds, inparticular from chemical or combinatorial libraries (i.e., libraries ofcompounds that differ in sequence or size but that have the similarbuilding blocks) or by rational, directed or random drug design. See forexample, Maulik et al., supra.

In a molecular diversity strategy, large compound libraries aresynthesized, for example, from peptides, oligonucleotides, carbohydratesand/or synthetic organic molecules, using biological, enzymatic and/orchemical approaches. The critical parameters in developing a moleculardiversity strategy include subunit diversity, molecular size, andlibrary diversity. The general goal of screening such libraries is toutilize sequential application of combinatorial selection to obtainhigh-affinity ligands for a desired target, and then to optimize thelead molecules by either random or directed design strategies. Methodsof molecular diversity are described in detail in Maulik, et al., ibid.

Maulik et al. also disclose, for example, methods of directed design, inwhich the user directs the process of creating novel molecules from afragment library of appropriately selected fragments; random design, inwhich the user uses a genetic or other algorithm to randomly mutatefragments and their combinations while simultaneously applying aselection criterion to evaluate the fitness of candidate ligands; and agrid-based approach in which the user calculates the interaction energybetween three dimensional receptor structures and small fragment probes,followed by linking together of favorable probe sites.

One embodiment of the present invention relates to isolated nucleic acidmolecules comprising, consisting essentially of, or consisting ofnucleic acid sequences that encode any of the proteins described herein,including a homologue or fragment of any of such proteins, as well asnucleic acid sequences that are fully complementary thereto. Inaccordance with the present invention, an isolated nucleic acid moleculeis a nucleic acid molecule that has been removed from its natural milieu(i.e., that has been subject to human manipulation), its natural milieubeing the genome or chromosome in which the nucleic acid molecule isfound in nature. As such, “isolated” does not necessarily reflect theextent to which the nucleic acid molecule has been purified, butindicates that the molecule does not include an entire genome or anentire chromosome in which the nucleic acid molecule is found in nature.An isolated nucleic acid molecule can include a gene. An isolatednucleic acid molecule that includes a gene is not a fragment of achromosome that includes such gene, but rather includes the codingregion and regulatory regions associated with the gene, but noadditional genes that are naturally found on the same chromosome. Anisolated nucleic acid molecule can also include a specified nucleic acidsequence flanked by (i.e., at the 5′ and/or the 3′ end of the sequence)additional nucleic acids that do not normally flank the specifiednucleic acid sequence in nature (i.e., heterologous sequences). Isolatednucleic acid molecule can include DNA, RNA (e.g., mRNA), or derivativesof either DNA or RNA (e.g., cDNA). Although the phrase “nucleic acidmolecule” primarily refers to the physical nucleic acid molecule and thephrase “nucleic acid sequence” primarily refers to the sequence ofnucleotides on the nucleic acid molecule, the two phrases can be usedinterchangeably, especially with respect to a nucleic acid molecule, ora nucleic acid sequence, being capable of encoding a protein or domainof a protein.

Preferably, an isolated nucleic acid molecule of the present inventionis produced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. Isolatednucleic acid molecules include natural nucleic acid molecules andhomologues thereof, including, but not limited to, natural allelicvariants and modified nucleic acid molecules in which nucleotides havebeen inserted, deleted, substituted, and/or inverted in such a mannerthat such modifications provide the desired effect on the biologicalactivity of the protein as described herein. Protein homologues (e.g.,proteins encoded by nucleic acid homologues) have been discussed indetail above.

A nucleic acid molecule homologue can be produced using a number ofmethods known to those skilled in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LabsPress, 1989). For example, nucleic acid molecules can be modified usinga variety of techniques including, but not limited to, classicmutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, PCR amplification and/ormutagenesis of selected regions of a nucleic acid sequence, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecule homologues can be selected from a mixture of modified nucleicacids by screening for the function of the protein encoded by thenucleic acid and/or by hybridization with a wild-type gene.

The minimum size of a nucleic acid molecule of the present invention isa size sufficient to encode a protein having the desired biologicalactivity, or sufficient to form a probe or oligonucleotide primer thatis capable of forming a stable hybrid with the complementary sequence ofa nucleic acid molecule encoding the natural protein (e.g., undermoderate, high or very high stringency conditions). As such, the size ofthe nucleic acid molecule encoding such a protein can be dependent onnucleic acid composition and percent homology or identity between thenucleic acid molecule and complementary sequence as well as uponhybridization conditions per se (e.g., temperature, salt concentration,and formamide concentration). The minimal size of a nucleic acidmolecule that is used as an oligonucleotide primer or as a probe istypically at least about 12 to about 15 nucleotides in length if thenucleic acid molecules are GC-rich and at least about 15 to about 18bases in length if they are AT-rich. There is no limit, other than apractical limit, on the maximal size of a nucleic acid molecule of thepresent invention, in that the nucleic acid molecule can include aportion of a protein-encoding sequence or a nucleic acid sequenceencoding a full-length protein.

One embodiment of the present invention is a recombinant nucleic acidmolecule comprising an isolated nucleic acid molecule of the presentinvention. According to the present invention, a recombinant nucleicacid molecule includes at least one isolated nucleic acid molecule ofthe present invention that is linked to a heterologous nucleic acidsequence. Such a heterologous nucleic acid sequence is typically arecombinant nucleic acid vector (e.g., a recombinant vector) which issuitable for cloning, sequencing, and/or otherwise manipulating thenucleic acid molecule, such as by expressing and/or delivering thenucleic acid molecule into a host cell to form a recombinant cell. Sucha vector contains heterologous nucleic acid sequences, that is nucleicacid sequences that are not naturally found adjacent to nucleic acidmolecules of the present invention, although the vector can also containregulatory nucleic acid sequences (e.g., promoters, untranslatedregions) which are naturally found adjacent to nucleic acid molecules ofthe present invention (discussed in detail below). The vector can beeither RNA or DNA, either prokaryotic or eukaryotic, and typically is avirus or a plasmid. The vector can be maintained as an extrachromosomalelement (e.g., a plasmid) or it can be integrated into the chromosome.The entire vector can remain in place within a host cell, or undercertain conditions, the plasmid DNA can be deleted, leaving behind thenucleic acid molecule of the present invention. The integrated nucleicacid molecule can be under chromosomal promoter control, under native orplasmid promoter control, or under a combination of several promotercontrols. Single or multiple copies of the nucleic acid molecule can beintegrated into the chromosome. As used herein, the phrase “recombinantnucleic acid molecule” is used primarily to refer to a recombinantvector into which has been ligated the nucleic acid sequence to becloned, manipulated, transformed into the host cell (i.e., the insert).

In one embodiment, a recombinant vector of the present invention is anexpression vector, such that the recombinant nucleic molecule producedby inserting a nucleic acid molecule into the vector can be used toexpress, or produce the protein encoded by the nucleic acid moleculeinsert. As used herein, the phrase “expression vector” is used to referto a vector that is suitable for production of an encoded product (e.g.,a protein of interest). More particularly, a nucleic acid sequenceencoding the product to be produced is inserted into the recombinantvector to produce a recombinant nucleic acid molecule. The nucleic acidsequence encoding the protein to be produced is inserted into the vectorin a manner that operatively links the nucleic acid sequence toregulatory sequences in the vector (e.g., expression control sequences)which enable the transcription and translation of the nucleic acidsequence when the recombinant molecule is introduced into a host cell.

In another embodiment, a recombinant vector used in a recombinantnucleic acid molecule of the present invention is a targeting vector. Asused herein, the phrase “targeting vector” is used to refer to a vectorthat is used to deliver a particular nucleic acid molecule into arecombinant host cell, wherein the nucleic acid molecule is used todelete or inactivate an endogenous gene within the host cell (i.e., usedfor targeted gene disruption or knock-out technology) and/or to insertor replace a new, exogenous gene or nucleic acid molecule into thegenome of the host cell.

According to the present invention, the phrase “operatively linked”refers to linking a nucleic acid molecule to an expression controlsequence (e.g., a transcription control sequence and/or a translationcontrol sequence) in a manner such that the molecule can be expressedwhen transfected (i.e., transformed, transduced, transfected, conjugatedor conduced) into a host cell. Transcription control sequences aresequences that control the initiation, elongation, or termination oftranscription. Particularly important transcription control sequencesare those that control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in a host cell into which the recombinant nucleic acid moleculeis to be introduced.

Recombinant molecules of the present invention, which can be either DNAor RNA, can also contain additional regulatory sequences, such astranslation regulatory sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell. Inone embodiment, a recombinant molecule of the present invention,including those which are integrated into the host cell chromosome, alsocontains secretory signals (i.e., signal segment nucleic acid sequences)to enable an expressed protein to be secreted from the cell thatproduces the protein. Suitable signal segments include a signal segmentthat is naturally associated with a protein of the present invention orany heterologous signal segment capable of directing the secretion of aprotein according to the present invention.

One or more recombinant molecules of the present invention can be usedto produce an encoded product of the present invention. In oneembodiment, an encoded product is produced by expressing a nucleic acidmolecule as described herein under conditions effective to produce theprotein. A preferred method to produce an encoded protein is bytransfecting a host cell with one or more recombinant molecules to forma recombinant cell. Suitable host cells to transfect include, but arenot limited to, any bacterial, fungal (e.g., yeast), insect, plant oranimal cell that can be transfected. Host cells can be eitheruntransfected cells or cells that are already transfected with at leastone nucleic acid molecule.

According to the present invention, the term “transfection” is used torefer to any method by which an exogenous nucleic acid molecule (i.e., arecombinant nucleic acid molecule) can be inserted into the cell. Theterm “transformation” can be used interchangeably with the term“transfection” when such term is used to refer to the introduction ofnucleic acid molecules into microbial cells, such as bacteria and yeast.In microbial systems, the term “transformation” is used to describe aninherited change due to the acquisition of exogenous nucleic acids bythe microorganism and is essentially synonymous with the term“transfection”. However, in animal cells, transformation has acquired asecond meaning which can refer to changes in the growth properties ofcells in culture after they become cancerous, for example. Therefore, toavoid confusion, the term “transfection” is preferably used with regardto the introduction of exogenous nucleic acids into animal cells, andthe term “transfection” will be used herein to generally encompass bothtransfection of animal cells and transformation of microbial cells, tothe extent that the terms pertain to the introduction of exogenousnucleic acids into a cell. Therefore, transfection techniques include,but are not limited to, transformation, electroporation, microinjection,lipofection, adsorption, infection and protoplast fusion.

In one embodiment, a protein of the present invention is produced byculturing a cell that expresses the protein under conditions effectiveto produce the protein, and recovering the protein. A preferred cell toculture is a recombinant cell of the present invention. Effectiveculture conditions include, but are not limited to, effective media,bioreactor, temperature, pH and oxygen conditions that permit proteinproduction. An effective medium refers to any medium in which a cell iscultured to produce a protein of the present invention. Such mediumtypically comprises an aqueous medium having assimilable carbon,nitrogen and phosphate sources, and appropriate salts, minerals, metalsand other nutrients, such as vitamins. Examples of suitable media andculture conditions are discussed in detail in the Examples section.Cells of the present invention can be cultured in conventionalfermentation bioreactors, shake flasks, test tubes, microtiter dishes,and petri plates. Culturing can be carried out at a temperature, pH andoxygen content appropriate for a recombinant cell. Such culturingconditions are within the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell; be secreted into the culture medium; be secreted intoa space between two cellular membranes, such as the periplasmic space inE. coli; or be retained on the outer surface of a cell or viralmembrane. The phrase “recovering the protein” refers to collecting thewhole culture medium containing the protein and need not implyadditional steps of separation or purification. Proteins of the presentinvention can be purified using a variety of standard proteinpurification techniques, such as, but not limited to, affinitychromatography, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.

Proteins of the present invention are preferably retrieved, obtained,and/or used in “substantially pure” form. As used herein, “substantiallypure” refers to a purity that allows for the effective use of theprotein in vitro, ex vivo or in vivo according to the present invention.For a protein to be useful in an in vitro, ex vivo or in vivo methodaccording to the present invention, it is substantially free ofcontaminants, other proteins and/or chemicals that might interfere orthat would interfere with its use in a method disclosed by the presentinvention, or that at least would be undesirable for inclusion with theprotein when it is used in a method disclosed by the present invention.Such methods include antibody production, agonist/antagonistidentification assays, preparation of therapeutic compositions,administration in a therapeutic composition, and all other methodsdisclosed herein. Preferably, a “substantially pure” protein, asreferenced herein, is a protein that can be produced by any method(i.e., by direct purification from a natural source, recombinantly, orsynthetically), and that has been purified from other protein componentssuch that the protein comprises at least about 80% weight/weight of thetotal protein in a given composition, and more preferably, at leastabout 85%, and more preferably at least about 90%, and more preferablyat least about 91%, and more preferably at least about 92%, and morepreferably at least about 93%, and more preferably at least about 94%,and more preferably at least about 95%, and more preferably at leastabout 96%, and more preferably at least about 97%, and more preferablyat least about 98%, and more preferably at least about 99%,weight/weight of the total protein in a given composition.

One embodiment of the present invention relates to an antibody orantigen binding fragment that selectively binds to a protein of thepresent invention (e.g., Cystatin C protein). Such an antibody canselectively bind to any of the described herein, including fragments andother homologues of such receptors. According to the present invention,the phrase “selectively binds to” refers to the ability of an antibody,antigen binding fragment or binding partner of the present invention topreferentially bind to specified proteins. More specifically, the phrase“selectively binds” refers to the specific binding of one protein toanother (e.g., an antibody, fragment thereof, or binding partner to anantigen), wherein the level of binding, as measured by any standardassay (e.g., an immunoassay), is statistically significantly higher thanthe background control for the assay. For example, when performing animmunoassay, controls typically include a reaction well/tube thatcontain antibody or antigen binding fragment alone (i.e., in the absenceof antigen), wherein an amount of reactivity (e.g., non-specific bindingto the well) by the antibody or antigen binding fragment thereof in theabsence of the antigen is considered to be background. Binding can bemeasured using a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immunoblot assays, etc.

In one embodiment, the antibody is a bi- or multi-specific antibody. Abi-specific (or multi-specific) antibody is capable of binding two (ormore) antigens, as with a divalent (or multivalent) antibody, but inthis case, the antigens are different antigens (i.e., the antibodyexhibits dual or greater specificity). A bi-specific antibody suitablefor use in the present method includes an antibody having: (a) a firstportion (e.g., a first antigen binding portion) which binds to a givenprotein; and (b) a second portion which binds to a cell surface moleculeexpressed by a cell which expresses the protein. In this embodiment, thesecond portion can bind to any cell surface molecule. In a preferredembodiment, the second portion is capable of targeting the regulatoryantibody to a specific target cell (i.e., the regulatory antibody bindsto a target molecule). For example, the second portion of thebi-specific antibody can be an antibody that binds to another cellsurface molecule on a target cell, such as a tumor cell.

Isolated antibodies of the present invention can include serumcontaining such antibodies, or antibodies that have been purified tovarying degrees. Whole antibodies of the present invention can bepolyclonal or monoclonal. Alternatively, functional equivalents of wholeantibodies, such as antigen binding fragments in which one or moreantibody domains are truncated or absent (e.g., Fv, Fab, Fab′, or F(ab)₂fragments), as well as genetically-engineered antibodies or antigenbinding fragments thereof, including single chain antibodies orantibodies that can bind to more than one epitope (e.g., bi-specificantibodies), or antibodies that can bind to one or more differentantigens (e.g., bi- or multi-specific antibodies), may also be employedin the invention.

The invention also extends to non-antibody polypeptides, sometimesreferred to as binding partners, that have been designed to bindspecifically to, and either activate or inhibit as appropriate, a CystCprotein or another protein (e.g., a TβRII). Examples of the design ofsuch polypeptides, which possess a prescribed ligand specificity aregiven in Beste et al. (Proc. Natl. Acad. Sci. 96:1898-1903, 1999),incorporated herein by reference in its entirety.

One embodiment of the present invention includes an antibody,antigen-binding fragment thereof, or binding partner that binds to CystCand inhibits at least one biological activity of the CystC. For example,a preferred embodiment of the invention comprises an antibody,antigen-binding fragment thereof, or binding partner that selectivelybinds to CystC (e.g., in the C-terminal region of CystC), and inhibitsthe TGF-β-regulating portion of CystC, but not the cysteine proteaseinhibitory activity of CystC. The opposite embodiment (inhibition of thecysteine protease inhibitory activity but not the TGF-β regulatoryactivity) is also encompassed.

Another embodiment of the invention includes any CystC antagonist orinhibitor of CystC that results in a decrease in at least one biologicalactivity of CystC as described herein. Such inhibitors include, but arenot limited to, antibodies, antigen-binding fragments, binding partners,or other compounds (all described above) that selectively bind to andblock or inhibit at least one biological activity of Cyst C (e.g.,ability to bind to a TGF-β receptor, ability to activate a TGF-βreceptor, ability to inhibit a cysteine protease, etc.); soluble TGF-βreceptors and homologues thereof; CystC antagonists (including proteinand non-protein homologues or mimetics) that are competitive inhibitorsof natural CystC; and compounds that inhibit the expression of CystC. Inone embodiment, a soluble TGF-β receptor is preferably a soluble TβRIIreceptor, and more preferably, a soluble TβRII receptor that has beenmodified to have decreased binding affinity for TGF-β and normal orincreased binding affinity for CystC. Soluble TβRII are known in the art(e.g., see Examples), and can be modified based on the informationprovided herein to have the desired binding activity.

Some embodiments of the present invention include a composition orformulation comprising CystC or a fragment or homologue thereof(including agonists, antagonists, and other mimetics) or a regulatorthereof for diagnostic, screening or therapeutic purposes. Therefore,another embodiment of the invention relates to a composition comprisinga compound selected from: (i) an isolated CystC protein, fragment orhomologue thereof (including agonists and antagonists that areproteins); (ii) a CystC agonist or antagonist compound other than aprotein CystC homologue (e.g., a product of drug design); (iii) anisolated nucleic acid sequence encoding CystC or a homologue thereof, or(iv) a compound that affects the expression of an endogenous CystC genein a cell. The composition typically also includes a pharmaceuticallyacceptable carrier. In this aspect of the present invention, an isolatedCystC protein can be any of the CystC proteins previously describedherein, including, but not limited to, a wild-type CystC protein, aCystC protein homologue, a fragment of CystC and/or a CystC fusionprotein. Agonists and antagonists of CystC have also been describedabove. Isolated nucleic acid molecules encoding CystC or a homologue,fragment or fusion protein of CystC have also been described. In oneembodiment, a composition of the present invention includes acombination of at least two of any of the above-identified compounds.The compositions and their components can be used in any of thediagnostic or therapeutic embodiments of the invention described herein.

A composition, and particularly a therapeutic composition, of thepresent invention generally includes a carrier, and preferably, apharmaceutically acceptable carrier. According to the present invention,a “pharmaceutically acceptable carrier” includes pharmaceuticallyacceptable excipients and/or pharmaceutically acceptable deliveryvehicles, which are suitable for use in administration of thecomposition to a suitable in vitro, ex vivo or in vivo site. Preferredpharmaceutically acceptable carriers are capable of maintaining aprotein, compound, or nucleic acid molecule according to the presentinvention in a form that, upon arrival of the protein, compound, ornucleic acid molecule at the cell target in a culture or in patient, theprotein, compound or nucleic acid molecule is capable of interactingwith its target.

Suitable excipients of the present invention include excipients orformularies that transport or help transport, but do not specificallytarget a composition to a cell (also referred to herein as non-targetingcarriers). Examples of pharmaceutically acceptable excipients include,but are not limited to water, phosphate buffered saline, Ringer'ssolution, dextrose solution, serum-containing solutions, Hank'ssolution, other aqueous physiologically balanced solutions, oils, estersand glycols. Aqueous carriers can contain suitable auxiliary substancesrequired to approximate the physiological conditions of the recipient,for example, by enhancing chemical stability and isotonicity.

Suitable auxiliary substances include, for example, sodium acetate,sodium chloride, sodium lactate, potassium chloride, calcium chloride,and other substances used to produce phosphate buffer, Tris buffer, andbicarbonate buffer. Auxiliary substances can also include preservatives,such as thimerosal, m- or o-cresol, formalin and benzol alcohol.Compositions of the present invention can be sterilized by conventionalmethods and/or lyophilized.

One type of pharmaceutically acceptable carrier includes a controlledrelease formulation that is capable of slowly releasing a composition ofthe present invention into a patient or culture. As used herein, acontrolled release formulation comprises a compound of the presentinvention (e.g., a protein (including homologues), a drug, an antibody,a nucleic acid molecule, or a mimetic) in a controlled release vehicle.Suitable controlled release vehicles include, but are not limited to,biocompatible polymers, other polymeric matrices, capsules,microcapsules, microparticles, bolus preparations, osmotic pumps,diffusion devices, liposomes, lipospheres, and transdermal deliverysystems. Other carriers of the present invention include liquids that,upon administration to a patient, form a solid or a gel in situ.Preferred carriers are also biodegradable (i.e., bioerodible). When thecompound is a recombinant nucleic acid molecule, suitable deliveryvehicles include, but are not limited to liposomes, viral vectors orother delivery vehicles, including ribozymes. Natural lipid-containingdelivery vehicles include cells and cellular membranes. Artificiallipid-containing delivery vehicles include liposomes and micelles. Adelivery vehicle of the present invention can be modified to target to aparticular site in a patient, thereby targeting and making use of acompound of the present invention at that site. Suitable modificationsinclude manipulating the chemical formula of the lipid portion of thedelivery vehicle and/or introducing into the vehicle a targeting agentcapable of specifically targeting a delivery vehicle to a preferredsite, for example, a preferred cell type. Other suitable deliveryvehicles include gold particles, poly-L-lysine/DNA-molecular conjugates,and artificial chromosomes.

A pharmaceutically acceptable carrier which is capable of targeting isherein referred to as a “delivery vehicle.” Delivery vehicles of thepresent invention are capable of delivering a composition of the presentinvention to a target site in a patient. A “target site” refers to asite in a patient to which one desires to deliver a composition. Forexample, a target site can be any cell which is targeted by directinjection or delivery using liposomes, viral vectors or other deliveryvehicles, including ribozymes and antibodies. Examples of deliveryvehicles include, but are not limited to, artificial and naturallipid-containing delivery vehicles, viral vectors, and ribozymes.Natural lipid-containing delivery vehicles include cells and cellularmembranes. Artificial lipid-containing delivery vehicles includeliposomes and micelles. A delivery vehicle of the present invention canbe modified to target to a particular site in a subject, therebytargeting and making use of a compound of the present invention at thatsite. Suitable modifications include manipulating the chemical formulaof the lipid portion of the delivery vehicle and/or introducing into thevehicle a compound capable of specifically targeting a delivery vehicleto a preferred site, for example, a preferred cell type, such as a tumorcell. Specifically, targeting refers to causing a delivery vehicle tobind to a particular cell by the interaction of the compound in thevehicle to a molecule on the surface of the cell. Suitable targetingcompounds include ligands capable of selectively (i.e., specifically)binding another molecule at a particular site. Examples of such ligandsinclude antibodies, antigens, receptors and receptor ligands.Manipulating the chemical formula of the lipid portion of the deliveryvehicle can modulate the extracellular or intracellular targeting of thedelivery vehicle. For example, a chemical can be added to the lipidformula of a liposome that alters the charge of the lipid bilayer of theliposome so that the liposome fuses with particular cells havingparticular charge characteristics.

Another delivery vehicle comprises a viral vector. A viral vectorincludes an isolated nucleic acid molecule useful in the presentinvention, in which the nucleic acid molecules are packaged in a viralcoat that allows entrance of DNA into a cell. A number of viral vectorscan be used, including, but not limited to, those based on alphaviruses,poxviruses, adenoviruses, herpesviruses, lentiviruses, adeno-associatedviruses and retroviruses.

One embodiment of the present invention is to use the CystC proteins,homologues, mimetics and related TGF-β-regulatory compounds describedherein, either alone, or in a composition for the regulation of TGF-βactivity and/or the regulation of cathepsin B activity (including theregulation of TGF-β by cathepsin B). Regulation of TGF-β activity caninclude inhibition of TGF-β activity and enhancement of TGF-β activity.In embodiments where TGF-β activity is inhibited, such methods can beextended to methods to inhibit tumor malignancy (including metastaticmalignancy) or a proliferative or fibrotic condition or disease, andparticularly tumor malignancies or proliferative or fibrotic conditionsor diseases that are mediated at least in part by TGF-β expression oractivity, and/or by cathepsin-B regulation of TGF-β expression oractivity. Methods involving the inhibition of TGF-β activity generallycomprise administering to a patient or alternatively, contacting a cell(isolated or in vivo), with Cystatin C or a homologue or syntheticmimetic thereof having Cystatin C biological activity, or with acomposition comprising such agents. When the cell is a tumor cell or thepatient has cancer, the agent preferably is administered in an amounteffective to inhibit the biological activity of cathepsin B and/or TGF-βactivity in the tumor cell or in the microenvironment of the tumor cell.In one embodiment, the agent inhibits the biological activity ofextracellular cathepsin B. In embodiments where the TGF-β inhibitoryactivity of CystC is to be decreased, the method includes administeringto a patient or cell an inhibitor of CystC or Cyst antagonist, asdescribed above.

In a related embodiment, the invention includes a method to increase theexpression and/or biological activity of endogenous Cystatin C in a hostcell, and particularly, a tumor cell. This method includes the step ofadministering a compound or composition that increases the expression oractivity of CystC that is endogenously expressed by a host cell. Suchcompounds can be identified using the methods of identification ofregulatory compounds described below. Another embodiment includes amethod to decrease the expression and/or biological activity ofendogenous CystC in a host cell, which includes administering a compoundor composition that decreases the expression or activity of endogenousCystC.

A composition which includes a regulatory compound or agent or proteinof the invention can be delivered to a cell culture or patient by anysuitable method. Selection of such a method will vary with the type ofcompound being administered or delivered (i.e., protein, nucleic acid,mimetic), the mode of delivery (i.e., in vitro, in vivo, ex vivo) andthe goal to be achieved by administration/delivery of the compound orcomposition. According to the present invention, an effectiveadministration protocol (i.e., administering a composition in aneffective manner) comprises suitable dose parameters and modes ofadministration that result in delivery of a composition to a desiredsite.

Administration routes include in vivo, in vitro and ex vivo routes. Invivo routes include, but are not limited to, intravenous administration,intraperitoneal administration, intramuscular administration, intranodaladministration, intracoronary administration, intraarterialadministration (e.g., into a carotid artery), subcutaneousadministration, transdermal delivery, intratracheal administration,subcutaneous administration, intraarticular administration,intraventricular administration, inhalation (e.g., aerosol),intracranial, intraspinal, intraocular, aural, intranasal, oral,pulmonary administration, impregnation of a catheter, and directinjection into a tissue. In a preferred embodiment of the presentinvention, a composition is administered by a parenteral route (e.g.,subcutaneous, intradermal, intravenous, intramuscular andintraperitoneal routes). Intravenous, intraperitoneal, intradermal,subcutaneous and intramuscular administrations can be performed usingmethods standard in the art. Aural delivery can include ear drops,intranasal delivery can include nose drops or intranasal injection, andintraocular delivery can include eye drops. Aerosol (inhalation)delivery can also be performed using methods standard in the art (see,for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference in itsentirety). Oral delivery can be performed by complexing a therapeuticcomposition of the present invention to a carrier capable ofwithstanding degradation by digestive enzymes in the gut of an animal.Examples of such carriers, include plastic capsules or tablets, such asthose known in the art. Direct injection techniques are particularlyuseful for suppressing graft rejection by, for example, injecting thecomposition into the transplanted tissue, or for site-specificadministration of a compound, such as at the site of a tumor.

Ex vivo refers to performing part of the regulatory step outside of thepatient, such as by transfecting a population of cells removed from apatient with a recombinant molecule comprising a nucleic acid sequenceencoding a protein according to the present invention under conditionssuch that the recombinant molecule is subsequently expressed by thetransfected cell, and returning the transfected cells to the patient. Invitro and ex vivo routes of administration of a composition to a cultureof host cells can be accomplished by a method including, but not limitedto, transfection, transformation, electroporation, microinjection,lipofection, adsorption, protoplast fusion, use of protein carryingagents, use of ion carrying agents, use of detergents for cellpermeabilization, and simply mixing (e.g., combining) a compound inculture with a target cell and/or target protein.

Various methods of administration and delivery vehicles disclosed hereinhave been shown to be effective for delivery of a nucleic acid moleculeto a target cell, whereby the nucleic acid molecule transfected the celland was expressed. In many studies, successful delivery and expressionof a heterologous gene was achieved in preferred cell types and/or usingpreferred delivery vehicles and routes of administration of the presentinvention.

For example, using liposome delivery, U.S. Pat. No. 5,705,151, issuedJan. 6, 1998, to Dow et al. demonstrated the successful in vivointravenous delivery of a nucleic acid molecule encoding a superantigenand a nucleic acid molecule encoding a cytokine in a cationic liposomedelivery vehicle, whereby the encoded proteins were expressed in tissuesof the animal, and particularly in pulmonary tissues. In addition, Liuet al., Nature Biotechnology 15:167, 1997, demonstrated that intravenousdelivery of cholesterol-containing cationic liposomes containing genespreferentially targets pulmonary tissues and effectively mediatestransfer and expression of the genes in vivo. Several publications byDzau and collaborators demonstrate the successful in vivo delivery andexpression of a gene into cells of the heart, including cardiac myocytesand fibroblasts and vascular smooth muscle cells using both naked DNAand Hemagglutinating virus of Japan-liposome delivery, administered byboth incubation within the pericardium and infusion into a coronaryartery (intracoronary delivery) (See, for example, Aoki et al., 1997, J.Mol. Cell, Cardiol. 29:949-959; Kaneda et al., 1997, Ann N.Y. Acad. Sci.811:299-308; and von der Leyen et al., 1995, Proc Natl Acad Sci USA92:1137-1141).

Delivery of numerous nucleic acid sequences has been accomplished byadministration of viral vectors encoding the nucleic acid sequences.Using such vectors, successful delivery and expression has been achievedusing ex vivo delivery (See, of many examples, retroviral vector; Blaeseet al., 1995, Science 270:475-480; Bordignon et al., 1995, Science270:470-475), nasal administration (CFTR-adenovirus-associated vector),intracoronary administration (adenoviral vector and Hemagglutinatingvirus of Japan, see above), intravenous administration (adeno-associatedviral vector; Koeberl et al., 1997, Proc Natl Acad Sci USA94:1426-1431). A publication by Maurice et al. (1999, J. Clin. Invest.104:21-29) demonstrated that an adenoviral vector encoding aβ2-adrenergic receptor, administered by intracoronary delivery, resultedin diffuse multichamber myocardial expression of the gene in vivo, andsubsequent significant increases in hemodynamic function and otherimproved physiological parameters. Levine et al. describe in vitro, exvivo and in vivo delivery and expression of a gene to human adipocytesand rabbit adipocytes using an adenoviral vector and direct injection ofthe constructs into adipose tissue (Levine et al., 1998, J. Nutr. Sci.Vitaminol. 44:569-572).

In the area of neuronal gene delivery, multiple successful in vivo genetransfers have been reported. Millecamps et al. reported the targetingof adenoviral vectors to neurons using neuron restrictive enhancerelements placed upstream of the promoter for the transgene(phosphoglycerate promoter). Such vectors were administered to mice andrats intramuscularly and intracerebrally, respectively, resulting insuccessful neuronal-specific transfection and expression of thetransgene in vivo (Millecamps et al., 1999, Nat. Biotechnol.17:865-869). As discussed above, Bennett et al. reported the use ofadeno-associated viral vector to deliver and express a gene bysubretinal injection in the neural retina in vivo for greater than 1year (Bennett, 1999, ibid.).

Gene delivery to synovial lining cells and articular joints has hadsimilar successes. Oligino and colleagues report the use of a herpessimplex viral vector which is deficient for the immediate early genes,ICP4, 22 and 27, to deliver and express two different receptors insynovial lining cells in vivo (Oligino et al., 1999, Gene Ther.6:1713-1720). The herpes vectors were administered by intraarticularinjection. Kuboki et al. used adenoviral vector-mediated gene transferand intraarticular injection to successfully and specifically express agene in the temporomandibular joints of guinea pigs in vivo (Kuboki etal., 1999, Arch. Oral. Biol. 44:701-709). Apparailly and colleaguessystemically administered adenoviral vectors encoding IL-10 to mice anddemonstrated successful expression of the gene product and profoundtherapeutic effects in the treatment of experimentally induced arthritis(Apparailly et al., 1998, J. Immunol. 160:5213-5220). In another study,murine leukemia virus-based retroviral vector was used to deliver (byintraarticular injection) and express a human growth hormone gene bothex vivo and in vivo (Ghivizzani et al., 1997, Gene Ther. 4:977-982).This study showed that expression by in vivo gene transfer was at leastequivalent to that of the ex vivo gene transfer. As discussed above,Sawchuk et al. has reported successful in vivo adenoviral vectordelivery of a gene by intraarticular injection, and prolonged expressionof the gene in the synovium by pretreatment of the joint with anti-Tcell receptor monoclonal antibody (Sawchuk et al., 1996, ibid. Finally,it is noted that ex vivo gene transfer of human interleukin-1 receptorantagonist using a retrovirus has produced high level intraarticularexpression and therapeutic efficacy in treatment of arthritis, and isnow entering FDA approved human gene therapy trials (Evans and Robbins,1996, Curr. Opin. Rheumatol. 8:230-234). Therefore, the state of the artin gene therapy has led the FDA to consider human gene therapy anappropriate strategy for the treatment of at least arthritis. Takentogether, all of the above studies in gene therapy indicate thatdelivery and expression of a recombinant nucleic acid molecule accordingto the present invention is feasible.

Another method of delivery of recombinant molecules is in anon-targeting carrier (e.g., as “naked” DNA molecules, such as istaught, for example in Wolff et al., 1990, Science 247, 1465-1468). Suchrecombinant nucleic acid molecules are typically injected by direct orintramuscular administration. Recombinant nucleic acid molecules to beadministered by naked DNA administration include an isolated nucleicacid molecule of the present invention, and preferably includes arecombinant molecule of the present invention that preferably isreplication, or otherwise amplification, competent. A naked nucleic acidreagent of the present invention can comprise one or more nucleic acidmolecules of the present invention including a dicistronic recombinantmolecule. Naked nucleic acid delivery can include intramuscular,subcutaneous, intradermal, transdermal, intranasal and oral routes ofadministration, with direct injection into the target tissue being mostpreferred. A preferred single dose of a naked nucleic acid vaccineranges from about 1 nanogram (ng) to about 100 μg, depending on theroute of administration and/or method of delivery, as can be determinedby those skilled in the art. Suitable delivery methods include, forexample, by injection, as drops, aerosolized and/or topically. In oneembodiment, pure DNA constructs cover the surface of gold particles (1to 3 μm in diameter) and are propelled into skin cells or muscle with a“gene gun.”

In the method of the present invention, therapeutic compositions can beadministered to any member of the Vertebrate class, Mammalia, including,without limitation, primates, rodents, livestock and domestic pets.Preferred patients to protect are humans.

In accordance with the present invention, a suitable single dose size ofa compound or composition is a dose that is capable of regulating thedesired biological activity, when administered one or more times over asuitable time period. Doses can vary depending upon the goal of theadministration or the condition or the disease being treated.Preferably, a protein or antibody of the present invention isadministered in an amount that is between about 50 U/kg and about 15,000U/kg body weight of the patient. In another embodiment, a protein orantibody is administered in an amount that is between about 0.01 μg andabout 10 mg per kg body weight of the patient, and more preferably,between about 0.1 μg and about 100 μg per kg body weight of the patient.When the compound to be delivered is a nucleic acid molecule, anappropriate single dose results in at least about 1 pg of proteinexpressed per mg of total tissue protein per μg of nucleic aciddelivered. More preferably, an appropriate single dose is a dose whichresults in at least about 10 pg of protein expressed per mg of totaltissue protein per μg of nucleic acid delivered; and even morepreferably, at least about 50 pg of protein expressed per mg of totaltissue protein per μg of nucleic acid delivered; and most preferably, atleast about 100 pg of protein expressed per mg of total tissue proteinper μg of nucleic acid delivered. A preferred single dose of a nakednucleic acid vaccine ranges from about 1 nanogram (ng) to about 100 μg,depending on the route of administration and/or method of delivery, ascan be determined by those skilled in the art. Suitable delivery methodsinclude, for example, by injection, as drops, aerosolized and/ortopically. In one embodiment, pure DNA constructs cover the surface ofgold particles (1 to 3 μm in diameter) and are propelled into skin cellsor muscle with a “gene gun.” It will be obvious to one of skill in theart that the number of doses administered to a patient is dependent uponthe goal of the administration (e.g., the extent of the disease orcondition to be treated and the response of an individual patient to thetreatment).

As used herein, the phrase “protected from a disease” refers to reducingthe symptoms of the disease; reducing the occurrence of the disease,and/or reducing the severity of the disease. Protecting a patient canrefer to the ability of a composition of the present invention, whenadministered to a patient, to prevent a disease from occurring and/or tocure or to alleviate disease symptoms, signs or causes. As such, toprotect a patient from a disease includes both preventing diseaseoccurrence (prophylactic treatment) and treating a patient that has adisease (therapeutic treatment) to reduce the symptoms of the disease.In particular, protecting a patient from a disease or enhancing anothertherapy (e.g., transplantation) is accomplished by regulating a givenactivity such that a beneficial effect is obtained. A beneficial effectcan easily be assessed by one of ordinary skill in the art and/or by atrained clinician who is treating the patient. The term, “disease”refers to any deviation from the normal health of a mammal and includesa state when disease symptoms are present, as well as conditions inwhich a deviation (e.g., infection, gene mutation, genetic defect, etc.)has occurred, but symptoms are not yet manifested.

More specifically, a composition of the present invention comprisingCystC, a homologue or mimetic thereof, or other related compound, whenadministered to an animal by the method of the present invention,preferably produces a result which can include alleviation of thedisease (e.g., reduction of at least one symptom or clinicalmanifestation of the disease), elimination of the disease, reduction ofa tumor or lesion associated with the disease, elimination of a tumor orlesion associated with the disease, prevention or alleviation of asecondary disease resulting from the occurrence of a primary disease(e.g., metastatic cancer resulting from a primary cancer), prevention ofthe disease, and stimulation of effector cell immunity against thedisease. In particular, the compositions of the present invention areeffective to regulate TGF-β and/or cathepsin B activity and therebyregulate the downstream effects of such activities, and moreparticularly, to improve the therapeutic response to human malignancies,as well as a variety of proliferative and fibrotic diseases regulated byTGF-β.

Cancers to be treated or prevented using the methods and compositions ofthe present invention include, but are not limited to, melanomas,squamous cell carcinoma, breast cancers, head and neck carcinomas,thyroid carcinomas, soft tissue sarcomas, bone sarcomas, testicularcancers, prostatic cancers, ovarian cancers, bladder cancers, skincancers, brain cancers, angiosarcomas, hemangiosarcomas, mast celltumors, primary hepatic cancers, lung cancers, pancreatic cancers,gastrointestinal cancers, renal cell carcinomas, hematopoieticneoplasias, and metastatic cancers thereof. A therapeutic composition ofthe present invention is useful for inhibiting tumor malignancy byinhibiting (i) cathepsin B-mediated invasion, (ii) cathepsin B-mediatedTGF-β activation, and/or (iii) TGF-β signal transduction. Preferably,the use of CystC and related compounds according to the presentinvention in an animal that has or is at risk of developing cancerproduces a result selected from the group of alleviation of the cancer,prevention of metastatic cancer, or prevention of the primary cancer. Inone embodiment, the use of CystC and related compounds is particularlydesirable for the treatment of metastatic cancer or cancer that is laterstage cancer.

Proliferative or fibrotic conditions or diseases mediated at least inpart by TGF-β expression or activity that can be treated or preventedusing the methods and compositions of the present invention include, butare not limited to, fibrosis (kidney, liver, pulmonary, etc.), excessivewound healing or scarring, hypertension, and organ transplant rejection(i.e., high TGF-β levels have been associated with rejection).

Certain diseases and conditions have been associated with increasedlevels of CystC and such diseases and candidates may also be treated byregulating the activity of CystC as described herein. Such diseases,include, but are not limited to, neurodegenerative diseases, rheumatoidarthritis and conditions associated with persistent pain.

One embodiment of the present invention relates to a method to identifya regulatory compound that regulates TGF-β activity, and preferably,that is an antagonist of TGF-β. Most preferably, the compound has theability to inhibit the TGF-β stimulation of initiating metastaticevents, including decreased cell polarization, reduced cell-cellcontact, and elevated cell invasion and migration. The compound may alsohave the ability to regulate cathepsin B activity (e.g., a homologue ofCystC), but such activity is not required of the compound of the presentinvention.

In one aspect of this embodiment, the method includes the steps of: (a)designing or identifying a putative antagonist compound based on thestructure of Cystatin C (e.g., the primary or tertiary structure); (b)synthesizing the compound; and (c) selecting compounds from (b) thatinhibit the biological activity of TGF-β. The Cystatin C can includeCystatin C from any animal species, but is preferably human Cystatin C.In this embodiment, the structure of Cystatin C can include the linearor primary structure, as well as the tertiary structure. Human CystatinC has been crystallized and the structure determined, and the atomiccoordinates for the tertiary structure of Cystatin C is found in theProtein Database Accession No. 1G96 (described in and deposited byJanowski et al., Nat. Struct. Biol. 8(4):316-320, 2001), incorporatedherein by reference in its entirety. The following publications, each ofwhich is incorporated herein by reference in its entirety, also describethe structure of CystC or cystatins: Ekiel et al., J. Mol. Biol.271(2):266-277, 1997; Bode et al., EMBO J. 7(8):2593-2599, 1988; andKozak et al., Acta Crystallog. D Biol. Crystallog. 55(11):1939-1942,1999. The primary or linear sequence of CystC from various animalspecies have been disclosed and described above, including variousinformation regarding the structure-to-function relationship of thelinear sequence.

Having the atomic coordinates that define the tertiary structure of theCystC protein, one can produce a representation or model of the threedimensional structure of a CystC protein, such as a computer model. Acomputer model of the present invention can be produced using anysuitable software program, including, but not limited to, MOLSCRIPT 2.0(Avatar Software AB, Heleneborgsgatan 21C, SE-11731 Stockholm, Sweden),the graphical display program O (Jones et. al., Acta Crystallography,vol. A47, p. 110, 1991), the graphical display program GRASP, or thegraphical display program INSIGHT. Suitable computer hardware useful forproducing an image of the present invention are known to those of skillin the art (e.g., a Silicon Graphics Workstation).

As used herein, the term “model” refers to a representation in atangible medium of the three dimensional structure of a protein,polypeptide or peptide. For example, a model can be a representation ofthe three dimensional structure in an electronic file, on a computerscreen, on a piece of paper (i.e., on a two dimensional medium), and/oras a ball-and-stick figure. Physical three-dimensional models aretangible and include, but are not limited to, stick models andspace-filling models. One can image a model, for example, on a computerscreen by expressing (or representing) and manipulating the model on acomputer screen using appropriate computer hardware and softwaretechnology known to those skilled in the art (e.g., Evans andSutherland, Salt Lake City, Utah, and Biosym Technologies, San Diego,Calif.). Computer screen images and pictures of the model can bevisualized in a number of formats including space-fillingrepresentations, α carbon traces, ribbon diagrams and electron densitymaps.

A representation or model of the three dimensional structure of a CystCprotein can also be determined using techniques which include molecularreplacement or SIR/MIR (single/multiple isomorphous replacement).Methods of molecular replacement are generally known by those of skillin the art (generally described in Brunger, Meth. Enzym., vol. 276, pp.558-580, 1997; Navaza and Saludjian, Meth. Enzym., vol. 276, pp.581-594, 1997; Tong and Rossmann, Meth. Enzym., vol. 276, pp. 594-611,1997; and Bentley, Meth. Enzym., vol. 276, pp. 611-619, 1997, each ofwhich are incorporated by this reference herein in their entirety) andare performed in a software program including, for example, AmoRe (CCP4,Acta Cryst. D50, 760-763 (1994) or XPLOR. Briefly, X-ray diffractiondata is collected from the crystal of a crystallized target structure.The X-ray diffraction data is transformed to calculate a Pattersonfunction. The Patterson function of the crystallized target structure iscompared with a Patterson function calculated from a known structure(referred to herein as a search structure). The Patterson function ofthe crystallized target structure is rotated on the search structurePatterson function to determine the correct orientation of thecrystallized target structure in the crystal. The translation functionis then calculated to determine the location of the target structurewith respect to the crystal axes. Once the crystallized target structurehas been correctly positioned in the unit cell, initial phases for theexperimental data can be calculated. These phases are necessary forcalculation of an electron density map from which structural differencescan be observed and for refinement of the structure. Preferably, thestructural features (e.g., amino acid sequence, conserved di-sulfidebonds, and β-strands or β-sheets) of the search molecule are related tothe crystallized target structure.

The model of the tertiary structure of the CystC protein can then beused to design or identify candidate compounds that are antagonists ofTGF-β, for example, of that are predicted to have other CystC biologicalactivity (e.g., cysteine protease inhibitory activity). Such compoundscan be designed using structure based drug design, which refers to theprediction of a conformation of a peptide, polypeptide, protein, orconformational interaction between a peptide or polypeptide, and acompound, using the three dimensional structure of the peptide,polypeptide or protein. Typically, structure based drug design isperformed with a computer. For example, generally, for a protein toeffectively interact with (e.g., bind to) a compound, it is necessarythat the three dimensional structure of the compound assume a compatibleconformation that allows the compound to bind to the protein in such amanner that a desired result is obtained upon binding. Knowledge of thethree dimensional structure of the protein enables a skilled artisan todesign a compound having such compatible conformation, or to select sucha compound from available libraries of compounds.

The step of designing compounds can include creating a new chemicalcompound or searching databases of libraries of known compounds (e.g., acompound listed in a computational screening database containing threedimensional structures of known compounds). Designing can also beperformed by simulating chemical compounds having substitute moieties atcertain structural features. The step of designing can include selectinga chemical compound based on a known function of the compound. Apreferred step of designing comprises computational screening of one ormore databases of compounds in which the three dimensional structure ofthe compound is known and is interacted (e.g., docked, aligned, matched,interfaced) with the three dimensional structure of a TGF-β receptor,for example, by computer (e.g. as described by Humblet and Dunbar,Animal Reports in Medicinal Chemistry, vol. 28, pp. 275-283, 1993, MVenuti, ed., Academic Press). Methods to synthesize suitable chemicalcompounds are known to those of skill in the art and depend upon thestructure of the chemical being synthesized. Methods to evaluate thebioactivity of the synthesized compound depend upon the bioactivity ofthe compound (e.g., inhibitory or stimulatory) are described herein.

One embodiment of the present invention for structure based drug designcomprises identifying a chemical compound that complements the shape ofa CystC protein or a portion thereof (e.g., the portion that interactswith TGF-β receptors). Such method is referred to herein as a “geometricapproach”. In a geometric approach, the number of internal degrees offreedom (and the corresponding local minima in the molecularconformation space) is reduced by considering only the geometric(hard-sphere) interactions of two rigid bodies, where one body (theactive site) contains “pockets” or “grooves” that form binding sites forthe second body (the complementing molecule, such as a ligand).

The geometric approach is described by Kuntz et al., J. Mol. Biol., vol.161, p. 269, 1982, which is incorporated by this reference herein in itsentirety. The algorithm for chemical compound design can be implementedusing the software program DOCK Package, Version 1.0 (available from theRegents of the University of California). Pursuant to the Kuntzalgorithm, the shape of the cavity or groove on the surface of astructure at a binding site or interface is defined as a series ofoverlapping spheres of different radii. One or more extant databases ofcrystallographic data (e.g., the Cambridge Structural Database Systemmaintained by University Chemical Laboratory, Cambridge University,Lensfield Road, Cambridge CB2 1EW, U.K.) or the Protein Data Bankmaintained by Brookhaven National Laboratory, is then searched forchemical compounds that approximate the shape thus defined.

Chemical compounds identified by the geometric approach can be modifiedto satisfy criteria associated with chemical complementarity, such ashydrogen bonding, ionic interactions or Van der Waals interactions.

Preferably, a compound that is identified by the method of the presentinvention originates from a compound having chemical and/orstereochemical complementarity with CystC or a portion thereof. Suchcomplementarity is characteristic of a compound that matches thestructure of the protein either in shape or in distribution of chemicalgroups and binds to TGF-β receptor (e.g., TβRII) to inhibit TGF-βbinding and/or inhibit cellular signal transduction in a cell expressingthe TGF-β receptor. More preferably, a compound that binds to a ligandbinding site of the TGF-β receptor associates with an affinity of atleast about 10⁻⁶ M, and more preferably with an affinity of at leastabout 10⁻⁷M, and more preferably with an affinity of at least about 10⁻⁸M.

The compounds of the present invention can be synthesized from readilyavailable starting materials. Various substituents on the compounds ofthe present invention can be present in the starting compounds, added toany one of the intermediates or added after formation of the finalproducts by known methods of substitution or conversion reactions. Ifthe substituents themselves are reactive, then the substituents canthemselves be protected according to the techniques known in the art. Avariety of protecting groups are known in the art, and can be employed.Examples of many of the possible groups can be found in “ProtectiveGroups in Organic Synthesis” by T. W. Green, John Wiley and Sons, 1981,which is incorporated herein in its entirety. For example, nitro groupscan be added by nitration and the nitro group can be converted to othergroups, such as amino by reduction, and halogen by diazotization of theamino group and replacement of the diazo group with halogen. Acyl groupscan be added by Friedel-Crafts acylation. The acyl groups can then betransformed to the corresponding alkyl groups by various methods,including the Wolff-Kishner reduction and Clemmenson reduction. Aminogroups can be alkylated to form mono-and di-alkylamino groups; andmercapto and hydroxy groups can be alkylated to form correspondingethers. Primary alcohols can be oxidized by oxidizing agents known inthe art to form carboxylic acids or aldehydes, and secondary alcoholscan be oxidized to form ketones. Thus, substitution or alterationreactions can be employed to provide a variety of substituentsthroughout the molecule of the starting material, intermediates, or thefinal product, including isolated products.

Since the compounds of the present invention can have certainsubstituents which are necessarily present, the introduction of eachsubstituent is, of course, dependent on the specific substituentsinvolved and the chemistry necessary for their formation. Thus,consideration of how one substituent would be affected by a chemicalreaction when forming a second substituent would involve techniquesfamiliar to one of ordinary skill in the art. This would further bedependent upon the ring involved.

It is to be understood that the scope of this invention encompasses notonly the various isomers which may exist but also the various mixturesof isomers which may be formed.

If the compound of the present invention contains one or more chiralcenters, the compound can be synthesized enantioselectively or a mixtureof enantiomers and/or diastereomers can be prepared and separated. Theresolution of the compounds of the present invention, their startingmaterials and/or the intermediates may be carried out by knownprocedures, e.g., as described in the four volume compendium OpticalResolution Procedures for Chemical Compounds: Optical ResolutionInformation Center, Manhattan College, Riverdale, N.Y., and inEnantiomers, Racemates and Resolutions, Jean Jacques, Andre Collet andSamuel H. Wilen; John Wiley & Sons, Inc., New York, 1981, which areincorporated herein in their entirety. Basically, the resolution of thecompounds is based on the differences in the physical properties ofdiastereomers by attachment, either chemically or enzymatically, of anenantiomerically pure moiety results in forms that are separable byfractional crystallization, distillation or chromatography.

When the compounds of the present invention contain an olefin moiety andsuch olefin moiety can be either cis- or trans-configuration, thecompounds can be synthesized to produce cis- or trans-olefin,selectively, as the predominant products. Alternatively, the compoundcontaining an olefin moiety can be produced as a mixture of cis- andtrans-olefins and separated using known procedures, for example, bychromatography as described in W. K. Chan, et al., J. Am. Chem. Soc.,1974, 96, 3642, which is incorporated herein in its entirety.

In the final step of the method, compounds identified by thestructure-based design or identification can be evaluated forbioactivity, and particularly, the ability to regulate TGF-β activity,and most particularly, to antagonize the activity of TGF-β, using any ofthe methods described herein (above and below) and further include anyart known method of evaluating TGF-β activity. Such methods include, butare not limited to, detection of the ability of the compound to bind toTGF-β receptor, and particularly to TβRII, or detection of the abilityof the compound to inhibit TGF-β-responsive gene expression, or theability of the compound to inhibit TGF-β-mediated tumor cell malignancyand invasion.

In another aspect of this embodiment, the method of identifyingcompounds includes the steps of: (a) identifying proteins that arestructural homologues of Cystatin C; and (b) evaluating proteins from(a) that are capable of regulating the biological activity of TGF-β. Forexample, the ability of the protein of (a) to bind to a receptor forTGF-β, such as TβRII can be evaluated. Methods of producing andidentifying compounds that are structural homologues of CystC usingcomputer design and the geometric approach, for example, have beendescribed in detail above. Given the structural information providedherein and especially the structure-to-function information correlatingCystC with its biological activities, one of skill in the art will alsobe able to produce more simple protein homologues, including fragments,of CystC for use in these methods. Methods for producing proteinhomologues of CystC have been described in detail above, and can simplybe identified and designed by sequence analysis and selection ofappropriate modifications that can be introduced using conventionalmolecular techniques (e.g., recombinant production of a homologue orfragment).

In yet another aspect of this embodiment, the method includes the stepsof: (a) contacting a cell that expresses a TGF-β receptor and Cystatin Cwith a putative regulatory compound; (b) detecting the expression ofCystatin C in the cell; and (c) comparing the expression of Cystatin Cafter contact with the compound to the expression of Cystatin C beforecontact with the compound, wherein detection of a change in theexpression of Cystatin C in the cells after contact with the compound ascompared to before contact with the compound indicates that the compoundis a putative regulator of TGF-β and TGF-β signal transduction.Alternatively, one can contact a cell that expresses a TGF-β receptorwith TGF-β and a putative homologue or mimetic of CystC and determinewhether the putative homologue or mimetic of CystC regulates theactivation of the TGF-β receptor by TGF-β. Other variations andcombinations of TGF-β, its receptors, CystC and homologues thereof canbe used in similar assays to determine the ability of candidateregulatory compounds to regulate TGF-β activity in a manner commensuratewith the regulation of TGF-β activity by wild-type CystC. Putativeregulatory compounds can be added to the assay system prior to,simultaneously with, or after the addition or provision of othercompounds in the assay (e.g., cells, TGF-β, CystC, etc.). Theabove-described assays may also be performed using non-cell basedassays, where the ability of a regulatory compound to mimic thebiological activity of wild-type CystC and/or to specifically regulateTGF-β-mediated activities in a CystC-specific manner are evaluated(e.g., by contacting TGF-β and/or its receptor, or CystC and a TGF-βreceptor, with a putative regulatory compound and evaluating binding oranother activity).

As used herein, the term “putative” or “candidate” refers to compoundshaving an unknown regulatory activity, at least with respect to theability of such compounds to regulate expression of a gene or protein orthe biological activity of a protein as described herein. In the methodof identifying a regulatory compound according to the present invention,the method can be a cell-based assay, or non-cell-based assay. Inaccordance with the present invention, a cell-based assay is conductedunder conditions which are effective to screen for regulatory compoundsuseful in the method of the present invention. Effective conditionsinclude, but are not limited to, appropriate media, temperature, pH andoxygen conditions that permit cell growth.

In one embodiment, the conditions under which a protein according to thepresent invention is contacted with a putative regulatory compound, suchas by mixing, are conditions in which the protein is not stimulated(activated) if essentially no regulatory compound is present.

In an alternate embodiment, the conditions under which a proteinaccording to the present invention is contacted with a putativeregulatory compound, such as by mixing, are conditions in which theprotein is normally stimulated (activated) if essentially no regulatorycompound is present.

The present methods involve contacting cells with the compound beingtested for a sufficient time to allow for interaction, activation orinhibition of the protein by the compound. The period of contact withthe compound being tested can be varied depending on the result beingmeasured, and can be determined by one of skill in the art. For example,for binding assays, a shorter time of contact with the compound beingtested is typically suitable, than when activation is assessed. As usedherein, the term “contact period” refers to the time period during whichcells are in contact with the compound being tested. The term“incubation period” refers to the entire time during which cells areallowed to grow prior to evaluation, and can be inclusive of the contactperiod. Thus, the incubation period includes all of the contact periodand may include a further time period during which the compound beingtested is not present but during which growth is continuing (in the caseof a cell based assay) prior to scoring.

The assay of the present invention can also be a non-cell based assay.In this embodiment, the putative regulatory compound can be directlycontacted with an isolated protein, or a protein component (e.g., anisolated extracellular portion of a receptor, or soluble receptor), andthe ability of the putative regulatory compound to bind to the proteinor protein component can be evaluated, such as by an immunoassay orother binding assay. The assay can then include the step of furtheranalyzing whether putative regulatory compounds which bind to a portionof the protein are capable of increasing or decreasing the activity ofthe protein. Such further steps can be performed by cell-based assay, asdescribed above, or by non-cell-based assay.

Methods to evaluate any compounds identified by any of theabove-described methods for use in any of the methods of the inventionhave been discussed in detail elsewhere herein. Agonists and antagonistsidentified by the above methods or any other suitable method are usefulin a variety of therapeutic methods as described herein.

The following experimental results are provided for purposes ofillustration and are not intended to limit the scope of the invention.

EXAMPLES

The following Materials and Methods were used in Examples 1-7 below.

Human Cystatin C Plasmids

A retroviral CystC vector was synthesized by PCR amplifying thefull-length human CystC cDNA from EST 4183311. The resulting PCR productwas shuttled through the pcDNA3.1/Myc-His B vector (InVitrogen) at EcoRI (N-terminus) and Xho I (C-terminus) restriction sites to C-terminallytag human CystC with Myc- and (His)6-tags. Afterward, the resultingtagged CystC cDNA was PCR amplified using oligonucleotides containingEco RI (N-terminus) and Xho I (C-terminus) restriction sites, andsubsequently ligated into identical sites immediately upstream of theIRES in the bicistronic retroviral vector, pMSCV-IRES-GFP (42).

Synthesis of a retroviral Δ14CystC vector proceeded by PCR amplifyingthe pGEX-4T1-contained Δ14CystC cDNA insert (see below), which wasshuttled through the pSecTag B vector (InVitrogen) at Nco I (N-terminus)and Bgl II (C-terminus) restriction sites. This cloning stepC-terminally tagged the Δ14CystC cDNA with the Myc- and (His)6-tags, aswell as appended the Ig leader sequence to its N-terminus to permit itssecretion when expressed in mammalian cells. The resulting taggedΔ14CystC was PCR amplified using oligonucleotides containing Hpa I(N-terminus) and Eco RI (C-terminus) restriction sites that facilitatedligation of the PCR fragment into pMSCV-IRES-GFP. When expressed inmammalian cells, Δ14CystC protein was slightly larger than that of itscorresponding wild-type CystC due to additional N-terminal amino acidsappended to Δ14CystC by the pSecTag vector.

All CystC and Δ14CystC cDNA inserts were sequenced in their entirety onan Applied Biosystems 377A DNA sequencing machine.

Fusion Protein Construction and Purification

A CystC fusion protein was synthesized by PCR amplifying full-lengthhuman CystC cDNA (less its signal sequence) using oligonucleotidescontaining Eco RI (N-terminus) and Xho I (C-terminus) restriction sites.The resulting PCR fragment was subcloned into the C-terminus ofglutathione S-transferase encoded by the bacterial expression vector,pGEX-4T1 (Amersham Pharmacia Biotech). Site-directed mutagenesis ofGST-CystC to delete its conserved cysteine proteinase inhibitor motif(Δ14CystC; residues 80-93) was performed using the QuikChangesite-directed mutagenesis kit (Stratagene) according to themanufacturer's recommendations. All fusion cDNA inserts were sequencedin their entirety on an Applied Biosystems 377A DNA sequencing machine.

The expression and purification of various GST fusion proteins fromtransformed E. coli was as described previously (43).

Soluble TβR-II Plasmid

A soluble human TGF-β type II receptor (TβR-II) was synthesized by PCRamplifying the extracellular domain of TβR-II (sTβR-II; nucleotides72-516) using oligonucleotides containing Kpn I (N-terminus) and Not I(C-terminus) restriction sites. The resulting PCR product was ligatedinto corresponding sites in the pSecTag B vector, which C-terminallytagged the sTIβR-II cDNA with Myc- and (His)6-tags and appended the Igleader sequence at its N-terminus. The resulting sTβR-II was sequencedin its entirety on an Applied Biosystems 377A DNA sequencing machine.

Isolation and Identification of Murine CystC

Murine 3T3-L1 fibroblasts were cultured in 15-cm plates in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal bovine serumuntil attaining 90% confluency. The cells were then rendered quiescentby extensive washing in PBS, followed by incubation in serum-free DMEMfor 12 hr at 37° C. Quiescent 3T3-L1 cells were metabolically labeledwith [35S]methionine in the absence or presence of TGF-β1 (10 ng/ml) for12 hr at 37° C. Afterward, naive- and TGF-β-conditioned media werecollected (10 plates/condition), clarified by centrifugation, andconcentrated initially to ˜3 ml in an Amicon concentrator (1000 Da MWC)and ultimately via by trichloroacetic acid/deoxycholate precipitation.The resulting protein pellets were resuspended, neutralized, andprecipitated with acetone prior to their resuspension in isoelectricfocusing buffer (8M urea, 4% CHAPS, 10 mM DTT, and 0.2% 3-10ampholytes). Protein samples were applied to 11 cm pH 3-10 isoelectricfocusing strips and developed according to the manufacturer'srecommendations (3 strips/condition; Amersham Pharmacia Biotech). Theisoelectric focused proteins were then fractionated through 10%SDS-PAGE, and subsequently transferred electrophoretically toImmobilon-P (Millipore). Secreted proteins were visualized by Coomassiestaining and autoradiography of the dried membranes. A differentiallyexpressed ˜18 kDa protein that was evident in TGF-β-conditioned mediawas excised from the membranes and sequenced using the Sequelon-AAsequencing kit (ABI).

Northern Blotting

Quiescent 3T3-L1 cells were incubated in the absence or presence ofTGF-β1 (5 ng/mL) for 0-48 hr, and subsequently were harvested in RNAzolReagent (Tel-Test) to isolate total RNA. Ten μg of total RNA was thenelectrophoresed through 1.2% agarose/formaldehyde gels and transferredto nylon membrane. The immobilized RNA was probed with a 32P-labeledhuman CystC cDNA probe for 1 hr at 68° C. in ExpressHyb (Clontech).Afterward, the membrane was washed according to the manufacturer'sinstructions, and Cystatin C mRNA was visualized by autoradiography.

Tumor Array

The effects of tumorigenesis on CystC expression were examined byhybridizing a 32P-radiolabeled full-length human CystC cDNA probe to amatched human normal/tumor cDNA array according to the manufacturer'sinstructions (Clontech). CystC expression in normal and malignant humantissues was visualized by autoradiography. cDNA array was stripped andreprobed with a 32P-labeled ubiquitin cDNA probe provided by themanufacturer. The expression of CystC was normalized to that ofubiquitin, and the ratio of CystC expression between individual pairs ofnormal and tumor tissue was determined. A ratio of ≧2 or ≦0.5 wasconsidered significant.

Retroviral Infections

Control (i.e., pMSCV-IRES-GFP), CystC, or Δ14CystC retroviralsupernatants were produced by EcoPac2 retroviral packaging cells(Clontech) and used to infect murine 3T3-L1 fibroblasts and human HT1080fibrosarcoma cells as described previously (42). Forty-eight hrpost-infection, the highest 10% of GFP-expressing cells were collectedon a MoFlo cell sorter(Cytomation), and subsequently expanded to yieldstable populations of control, CystC-, or Δ14CystC-expressing cellshaving equivalent GFP levels at a positivity rate of >90%.

Western Blotting

Conditioned media (2 ml) of 3T3-L1 cells stably expressing GFP, CystC orΔ14Cyst C were collected, clarified by centrifugation, and concentratedby trichloroacetic acid/deoxycholate precipitation. The proteins werefractionated through 12% SDS-PAGE gel and subsequently transferredelectrophoretically to nitrocellulose. The membrane was probed withanti-CystC polyclonal antibodies (1:1000, Upstate Biotechnology), andthe resulting immunocomplexes were visualized by enhancedchemiluminescence.

Invasion Assays

The effect CystC and D14CystC on the invasion of HT1080 and 3T3-L1 cellswas determined as described previously (42). Briefly, upper chamberswere coated with 100 μl of diluted Matrigel (1:50 in serum-free media),which was allowed to evaporate to dryness overnight at room temperature.The following morning the Matrigel mixtures were rehydrated andsubsequently cultured with control-, CystC-, or D14CystC-expressingHT1080 or 3T3-L1 cells at a density of 100,000 cells/chamber. Cellularinvasion was stimulated by addition of 2% serum to the lower chambers.Forty-eight hr later, the cells were washed twice in ice-cold PBS andimmediately fixed for 15 min with 95% ethanol. Cells remaining in theupper chambers were removed with a cotton swab, whereas those remainingin the lower chamber were stained with crystal violet. Quantifyinginvading cells was determined through two independent measures, whichyielded identical results: (i) manual counting under a light microscope,and (ii) crystal violet dye extraction by incubation of the membranes in10% acetic acid, followed by spectrophotometry at 590 nm.

In some experiments, the effects of recombinant CystC and D14CystC on3T3-L1 cell invasion was examined. To do so, 3T3-L1 cells (100,000cells/chamber) were allowed to invade through Matrigel in the absence orpresence of 10 mg/ml of recombinant GST, GST-CystC, or D14CystC,together with or without 5 ng/ml of TGF-β1. All subsequent procedureswere performed as described above.

Luciferase Reporter Gene Assays

Analysis of luciferase activity driven by the synthetic p3TP reporter((26); generously provided by Dr. Joan Massague, Sloan Kettering) wasperformed as described previously (42). Briefly, control, CystC-, orΔ14CystC-expressing 3T3-L1 or HT1080 cells were cultured onto 24-wellplates at a density of 45,000 cells/well and allowed to adhereovernight. The cells were transiently transfected the following morningby overnight exposure to LT1 liposomes (Mirus) containing 300 ng/wellp3TP-luciferase and 100 ng/well of pCMV-β-gal. Afterward, the cells werewashed twice with PBS and stimulated overnight in serum-free media withincreasing concentrations of TGF-β1 as indicated. The following morning,luciferase and β-gal activities contained in detergent-solubilized cellextracts were determined.

In some experiments, the effects of recombinant CystC and D14CystC onp3TP-luciferase activity 3T3-L1 cells was determined. To do so, 3T3-L1cells were transiently transfected as above, and subsequently stimulatedwith TGF-β1 (5 ng/ml) in the absence or presence of 10 mg/ml ofrecombinant GST, GST-CystC, or D14CystC. All subsequent procedures wereperformed as described above.

Iodinated TGF-β1 Radioligand Binding and Cross-Linking Assay

Mink lung Mv1Lu epithelial cells were plated onto 6-well plates andgrown until 90% confluency. The radioligand binding and cross-linking ofiodinated TGF-β1 (200 pM) to Mv1Lu cells in the absence or presence ofincreasing concentration of recombinant GST-Jun(1-79) or -CystC wasperformed as described previously (44). Afterward, cytokine:receptorcomplexes contained in detergent-solubilized whole cell extracts wereisolated by immunoprecipitation with anti-TβR-II antibodies as describedpreviously (44). TGF-β1 bound to cell surface TβR-I, TβR-II, and TβR-IIIwas visualized by exposure of the dried gels to a phosphor screen, whichwere developed 1-3 d later on a Molecular Dynamic Typhoon Scanner. Totalbound TGF-β1 was defined as the sum of signal intensities for TGF-β1cross-linked to TβR-I, TβR-II, and TβR-III in each condition.

In Vitro TGF-β1 Binding Assay

Human kidney 293T cells were plated onto 6-well plates and allowed toadhere overnight. The cells were transiently transfected the followingmorning by overnight exposure to LT1 liposomes containing 1 μg/wellsTβR-II, which encodes for the extracellular domain of TβR-II that isMyc-His-tagged at its C-terminus, and subsequently were placed inserum-free DMEM for an additional 24 hr. Afterward, theconditioned-media were collected and cleared of cellular debris by amicrocentrifugation. The resulting clarified supernatants weresupplemented with [¹²⁵I]TGF-β1, together with or without recombinantCystC (10 μg/ml), and subsequently were tumbled for 2 hr at 4° C.Afterward, TGF-β1:sTβR-II complexes were isolated by immunoprecipitationwith monoclonal anti-Myc antibodies (1 μg/mL; Covance) and fractionatedthrough 12% SDS-PAGE. TGF-β1 bound to recombinant sTβR-II was visualizedby a 1-3 d exposure of the dried gels to a phosphor screen, whichsubsequently was developed on a Molecular Dynamic Typhoon Scanner.

Example 1

The following example demonstrates that TGF-β1 induces CystC expressionin 3T3-L1 cells.

TGF-β governs cell microenvironments by regulating fibroblast expressionand secretion of cytokines, growth factors, and ECM proteins that alterthe survival, proliferation, and motility of normal and cancer cells. Toidentify fibroblast secretory proteins whose expression are regulated byTGF-β, the inventor collected, concentrated, and fractionated by2D-electrophoresis proteins present in naive- and TGF-β-conditionedmedia of murine 3T3-L1 fibroblasts. Fractionated proteins wereimmobilized to Immobilon-P, and were visualized by Coomassie stainingand autoradiography. Differentially expressed proteins regulated byTGF-β were excised and subjected to Edman sequencing. The results showedthat a highly basic protein of ˜18 kDa was prominently induced by TGF-β(data not shown). Edman sequencing of this protein returned an aminoacid sequence of (NH2)-ATPKQGPR-(COOH) (positions 21-28 of SEQ ID NO:4),corresponding to residues 21-28 of murine CystC.

TGF-β previously has been shown to stimulate CystC transcript expressionin murine embryo cells (18). To confirm that CystC expression was indeedinduced by TGF-β and to establish the mechanism for this effect,northern blot analysis was performed on total RNA isolated fromTGF-β-treated 3T3-L1 cells. TGF-β stimulated 3T3-L1 cells to synthesizeCystC transcript in a time-dependent manner (data not shown).Collectively, these findings establish CystC as a novel gene target forTGF-β in 3T3-L1 cells, ultimately leading to increased production andsecretion of CystC protein.

Example 2

The following example shows that tumorigenesis alters CystC expressionin human tissues.

Altered CystC expression has been associated with the development ofhuman pathologies, particularly cancer (3). In order to identify humancancers potentially susceptible to altered CystC expression, aradiolabeled human CystC cDNA probe was hybridized to a membrane arrayedwith matched normal/tumor cDNAs generated from cancer patients (data notshown). CystC expression was normalized to that of ubiquitin andnormal:tumor tissue CystC expression rations were determined. Ratios ≧2or ≦0.5 were considered significant. Of the 68 patients surveyed, CystCexpression was altered in 65% (44/68) of the tumors, of which 84%(37/44) showed downregulation. Significantly attenuated CystC expressionwas especially evident in cancers of the stomach (100%; 8/8 cases),prostate (100%; 3/3 cases), uterus (71%; 5/7 cases), kidney (60%; 9/15cases), rectum (57%; 4/7 cases), and colon (55%; 6/11 cases). Moreimportantly, CystC expression was aberrant in 69% of metastatic humanmalignancies (15/25), of which 73% (11/15) showed downregulated CystCexpression. Taken together, these findings support the notion that CystCnormally functions to suppress tumor formation, as well as the processesof invasion and metastasis.

Example 3

The following example demonstrates that CystC inhibits cathepsinB-mediated invasion in HT1080 cells.

Cathepsin B is a lysosomal cysteine proteinase that functions inintracellular protein catabolism, as well as in bone resorption, hormoneactivation, and antigen processing (5). During tumorigenesis, cancercells express a cathepsin B splice variant whose protein product issecreted into the extracellular milieu (5) and localized to the leadingedge of invasive tumors (19), thereby promoting cancer cell invasion andmetastasis. However, recent evidence has questioned the role ofintracellular versus extracellular cathepsin B in promoting cancer cellinvasion (20). To distinguish between these two possibilities, humanHT1080 fibrosarcoma cells that stably express the murine ecotropicreceptor (21) were infected with control (i.e., GFP), CystC, or Δ14CystCretrovirus. Afterward, cells that expressed GFP were isolated by flowcytometry to establish stable polyclonal populations of control-,CystC-, and Δ14CystC-expressing HT1080 cells (data not shown). Theinventor chose to study HT1080 cells because they are highly invasive,and because they express large quantities of secreted cathepsin B andcomparably little CystC (22). Thus, if extracellular cathepsin Bmediates HT1080 cell invasion, it was hypothesized that overexpressionof secreted CystC would attenuate their invasion through syntheticbasement membranes.

Briefly, GFP-, CystC-, or Δ14CystC-expressing HT0180 cells were allowedto invade through Matrigel-coated membranes for 48 hr. Data shown inFIG. 1A are the mean (±SE) of three (Δ14CystC) and nine (GFP and CystC)independent experiments presented as the percent invasion relative toGFP-expressing HT1080 cells (***, p<0.05; Student's T-Test).

Accordingly, CystC expression significantly inhibited HT1080 cellinvasion through Matrigel matrices (FIG. 1A). In contrast, expression ofΔ14CystC, which lacks the cysteine inhibitor motif and thus is unable toinactivate cathepsin B (23), failed to affect HT1080 cell invasion (FIG.1A). Furthermore, treating HT1080 cells with the cell impermeablecathepsin B inhibitor II (Calbiochem) significantly reduced theirinvasion through Matrigel matrices (by 47.1±8.5%; n=3, p<0.05), whiletreatment with the cell permeable cathepsin B inhibitor, CA-074ME(Calbiochem), failed to effect their invasiveness (101.0±5.6% ofcontrol; n=3).

Taken together, these findings indicate that HT1080 cell invasion occursin part through the proteinase activities of extracellular, notlysosomal, cathepsin B. These findings also suggest that measuresdesigned to augment CystC concentrations within tumor microenvironmentswill negate the oncogenic effects of cathepsin B.

Example 4

The following example shows that CystC inhibits TGF-β-responsivereporter gene expression via a cathepsin B-independent mechanism inHT1080 cells.

During tumorigenesis, TGF-β is frequently converted from a suppressor toa promoter of cancer cell growth, invasion, and metastasis (17, 24).Stimulation of HT1080 cells with TGF-β had no effect on their invasionthrough Matrigel matrices, nor on their expression of CystC (data notshown). Thus, while TGF-β clearly regulates CystC expression in murineembryo (18) and 3T3-L1 (Example 1) cells, the coupling of TGF-β to CystCexpression in HT1080 cells appears dysregulated. CystC has also beenreported to stimulate DNA synthesis in normal and transformed murineSwiss 3T3 fibroblasts (10), and in rat mesangial cells (11), as well asinhibit melanoma cell motility (25). However, overexpression of CystC orΔ14CystC in HT1080 cells failed to effect their synthesis of DNA andmigration to fibronectin (data not shown).

Extracellular cathepsin B expression has been linked to the activationof latent TGF-β from inactive ECM depots (6, 7). The inventor thereforehypothesized that CystC expression might impact TGF-β signaling via acathepsin B-dependent mechanism. To test this hypothesis, changes inluciferase expression driven by the synthetic p3TP-luciferase reportergene (26) were measured in control- and CystC-expressing HT1080 cells.Briefly, GFP-, CystC-, or Δ14CystC-expressing HT0180 cells weretransiently transfected with p3TP-luciferase and pCMV-β-gal. Thetransfectants were stimulated with increasing concentrations of TGF-β1(0-5 ng/ml) as indicated, and subsequently processed to measureluciferase and β-gal activities. Data are the mean (±SE) luciferaseactivities of four independent experiments normalized to untreatedGFP-expressing cells.

As shown in FIG. 1B, CystC significantly inhibited TGF-β-stimulatedluciferase activity driven by the p3TP promoter. Surprisingly, Δ14CystCwas equally effective as CystC in inhibiting luciferase activitystimulated by TGF-β (FIG. 1B). Collectively, these findings identifyCystC as a novel antagonist of TGF-β signaling, doing so through acathepsin B-independent pathway.

Example 5

The following example shows that CystC inhibits tonic andTGF-β-stimulated invasion in 3T3-L1 cells.

CystC previously has been reported to regulate cell proliferation (10,11) and motility (25). Similar to HT1080 cells, the inventor found thatCystC failed to effect 3T3-L1 cell DNA synthesis and migration tofibronectin (data not shown). However, the inventor has found that3T3-L1 fibroblasts readily invade through Matrigel matrices, and thatTGF-β enhances their ability to do so (see below). To determine theeffects of CystC on the invasiveness of 3T3-L1 cells, stable polyclonalpopulations of 3T3-L1 cells expressing GFP, CystC, or Δ14CystC weregenerated by bicistronic retroviral infection. Briefly, murine 3T3-L1cells were infected with ecotropic retrovirus encoding either GFP (i.e.,control), CystC, or D14CystC. The infectants were FACS-sorted by GFPexpression (highest 10%) to yield stable polyclonal populations ofcontrol, CystC, and D14CystC-expressing 3T3-L1 cells having equivalentGFP expression levels at a positivity rate of ≧90%. Immunoblotting3T3-L1 cell conditioned-media with anti-CystC antibodies demonstratedthat 3T3-L1 cells transduced with CystC- or D14CystC-retrovirusesconstitutively secrete recombinant CystC proteins into the medium. Theresulting 3T3-L1 cell lines had purities 90% and expressed GFPindistinguishably (data not shown). Furthermore, 3T3-L1 cells infectedwith CystC or Δ14CystC retroviruses expressed and secreted high levelsof recombinant CystC protein into the media, while those infected withcontrol retrovirus (i.e., GFP) were negative for recombinant CystCexpression (data not shown).

GFP-, CystC-, or Δ14CystC-expressing 3T3-L1 cells were allowed to invadethrough Matrigel-coated filters in the absence or presence of TGF-β1 (5ng/ml) for 48 hr, and the results are shown in FIG. 2A. As expected,3T3-L1 cells readily invaded through Matrigel matrices when stimulatedby serum: this response was unaffected by Δ14CystC, but was inhibitedsignificantly by CystC (FIG. 2A). FIG. 2A also shows that 3T3-L1 cellstreated with TGF-β exhibited a trend towards enhanced invasion; however,TGF-β treatment in combination with serum induced significantly more3T3-L1 cell invasion than that by serum alone (FIG. 2A). Interestingly,CystC expression blocked both components of 3T3-L1 cell invasion,whereas Δ14CystC blocked only the TGF-β-dependent component (FIG. 2A).Data are the mean (±SE) of four independent experiments presented as thepercent invasion relative to GFP-expressing 3T3-L1 cells. TGF-β1significantly enhanced 3T3-L1 cell invasion (***, p<0.05; Student'sT-Test). CystC expression significantly inhibited tonic (*, p<0.05;Student's T-Test) and TGF-β1-stimulated (**, p<0.05; Student's T-Test)3T3-L1 cell invasion, while Δ14CystC expression only significantlyinhibited TGF-β-stimulated 3T3-L1 cell invasion (**, p<0.05; Student'sT-Test).

Moreover, the inhibitory effects of CystC on 3T3-L1 cell invasion couldbe recapitulated by addition of recombinant CystC fusion proteins.Control or TGF-β1-stimulated (5 ng/ml) 3T3-L1 cells were allowed toinvade through Matrigel-coated filters for 48 hr in the absence orpresence of recombinant (10 mg/ml) GST, GST-CystC, or GST-D14CystC asindicated in FIG. 2B. As shown in FIG. 2B, treatment of 3T3-L1 cellswith recombinant GST (10 mg/ml) failed to effect their invasion inducedby serum or serum:TGF-β. In contrast, recombinant CystC administrationblocked 3T3-L1 cell invasion stimulated by serum and serum:TGF-β,whereas recombinant Δ14CystC selectively blocked that by TGF-β (FIG.2B). Data are the mean (±SE) of three independent experiments presentedas the percent invasion relative to untreated 3T3-L1 cells. TGF-β1significantly enhanced 3T3-L1 cell invasion (*, p<0.05; Student'sT-Test). Recombinant CystC significantly inhibited tonic (#, p<0.05;Student's T-Test) and TGF-β1-stimulated (**, p<0.05; Student's T-Test)3T3-L1 cell invasion, while Δ14CystC expression only significantlyinhibited TGF-β-stimulated 3T3-L1 cell invasion (**, p<0.05; Student'sT-Test). Collectively, these findings indicate that 3T3-L1 cell invasionproceeds through cathepsin B-dependent and TGF-β-dependent pathways.Moreover, the present inventor's findings show that CystC abrogated bothpathways, while Δ14CystC blocked only the TGF-β-stimulated pathway.

Example 6

The following example demonstrates that CystC and ΔCystC inhibitTGF-β-stimulated reporter gene expression in 3T3-L1 cells.

The present inventor's findings that TGF-β signaling was inhibited byCystC (FIGS. 1 and 2) independent of its actions on cathepsin B leadthem to hypothesize CystC as a general antagonist of TGF-β signaling. Totest this hypothesis, changes in p3TP-driven luciferase activity weremonitored in control, CystC-, Δ14CystC-expressing cells before and aftertheir stimulation with TGF-β. Briefly, GFP-, CystC-, orΔ14CystC-expressing 3T3-L1 cells were transiently transfected withp3TP-luciferase and pCMV-β-gal, and subsequently stimulated withincreasing concentrations of TGF-β1 (0-5 ng/ml) as indicated in FIG. 3A.Afterward, luciferase and β-gal activities contained indetergent-solubilized cell extracts were measured. FIG. 3A shows thatexpression of either CystC or Δ14CystC significantly reducedTGF-β-stimulated luciferase activity as compared to control cells. Dataare the mean (±SE) luciferase activities of four independent experimentsnormalized to untreated GFP-expressing cells.

The transfectants were then stimulated with TGF-β1 (5 ng/ml) in theabsence or presence of recombinant (10 mg/ml) GST, GST-CystC, orGST-D14CystC as indicated in FIG. 3B. Similar to their effects on 3T3-L1cell invasion, recombinant CystC and Δ14CystC both significantlyinhibited luciferase activity stimulated by TGF-β in 3T3-L1 cells (FIG.3B). Data are the mean (±SE) luciferase activities of five independentexperiments normalized to untreated GFP-expressing cells. CystC andΔ14CystC significantly inhibited TGF-β-stimulated luciferase activitydriven by the synthetic p3TP promoter (***, p<0.05; Student's T-Test).

Taken together, these findings identify CystC as a novel antagonist ofTGF-β signaling, doing so through a cathepsin B-independent mechanism.

Example 7

The following example shows that CystC antagonizes TGF-β1 binding toTGF-β receptors.

The present inventor's findings thus far have identified CystC as anovel TGF-β antagonist (FIGS. 1-3). The ability of recombinant CystC(and Δ14CystC) to recapitulate the inhibitory effects of CystCexpression on TGF-β signaling lead the inventor to speculate that CystCinhibits TGF-β signaling by antagonizing the interaction between TGF-βand its receptors. This hypothesis seemed especially attractive giventhe fact that the Type 3 cystatin family member, fetuin (also known asα2-HS-glycoprotein), inhibits TGF-β signaling by physically interactingwith and preventing the binding of TGF-β to its receptors (27-29).

Mink lung Mv1Lu epithelial cells were incubated for 3 hr at 4° C. with[125I]TGF-β1 (200 pM) in the absence or presence of increasingconcentrations (0-10 μg/ml; 0, 0.5, 1, 5, 10) of recombinant Jun(1-79)or CystC. Cytokine:receptor complexes were cross-linked by addition ofdisuccinimidyl suberate, and subsequently isolated fromdetergent-solubilized whole cell extracts by immunoprecipitation withanti-TβR-II antibodies. Although co-immunoprecipitation and affinitypull-down assays both failed to demonstrate a direct physicalinteraction between CystC and TGF-β1 (data not shown), iodinated TGF-β1binding and cross-linking assays showed that recombinant CystCdose-dependently inhibited the binding of TGF-β to its cell surfacereceptors (FIG. 4A). The reduction in TGF-β1 binding was specific toCystC because increasing concentrations of recombinant Jun(1-79) had noeffect on the binding of iodinated TGF-β1 to its receptors (FIG. 4A).Data were generated based on a representative phosphor image ofiodinated TGF-β1 bound to TβR-I, TβR-II, and TβR-III. FIG. 4A depictsthe mean TGF-β1 (±SE) binding observed in six independent experimentsand is presented as the percent TGF-β1 binding normalized to that in theabsence of added fusion protein. This finding implicates CystC as anovel TGF-β receptor antagonist.

Transmembrane signaling by TGF-β commences by its binding to eitherTβR-III, which then associates with and binds to TβR-II, or directly toTβR-II, which then associates with and binds to the TβR-I (17, 30). Theexperiment above demonstrated that CystC equally reduced the binding ofTGF-β1 to all three of its cell surface receptors, which suggests thatCystC selectively antagonized initial TGF-β binding, not its subsequentreceptor multimerization. The inventor therefore hypothesized that CystCantagonized TGF-β binding to TβR-II. To test this hypothesis, an invitro TGF-β capture assay was performed that measured the effects ofCystC on the binding of iodinated TGF-β1 to soluble human TβR-II. ThisTβR-II construct was chosen for study because it binds TGF-β (31) andhas itself been used to antagonize TGF-β signaling (32-34). Briefly,human 293T cells were transiently transfected with the ligand bindingextracellular domain of TβR-II (sTβR-II), which is a soluble, secretedprotein. Conditioned-media from mock- or sTβR-II-transfected 293T cellswas collected and incubated for 2 hr at 4° C. with [125I]TGF-β1 (200 pM)in the absence or presence of recombinant CystC (10 μg/ml).Cytokine:receptor complexes were isolated by immunoprecipitation withanti-Myc antibodies. As expected, significantly more iodinated TGF-β1was captured by conditioned media containing sTβR-II than was capturedby control media (FIG. 4B). More importantly, recombinant CystCcompletely abrogated the binding of iodinated TGF-β1 to sTβR-II (FIG.4B). Upper panel shows a representative autoradiograph of iodinatedTGF-β1 bound to sTβR-II, while lower panel depicts the mean TGF-β1 (±SE)binding from three independent experiments presented as the percentTGF-β1 binding normalized to conditioned-media of mock transfected cellswithout added fusion protein. sTβR-II significantly enhanced TGF-β1capture from conditioned-media (*, p<0.05; Student's T-Test). Thebinding of TGF-β1 to sTβR-II was significantly reduced by recombinantCystC (**, p<0.05; Student's T-Test).

Collectively, these findings have identified CystC as a novel antagonistof TGF-β signaling, doing so by inhibiting TGF-β binding to TβR-II.

The following Materials and Methods were used in Examples 8-9 below.

Recombinant CystC Expression and Purification

The synthesis of bacterial expression vectors encoding human CystC orΔ14CystC fused to the C-terminus of GST, as well as their purificationfrom transformed E. coli was described in Examples 1-7 above.

Retroviral CystC Expression

The creation of bicistronic retroviral vectors (i.e., pMSCV-IRES-GFP)encoding human CystC or Δ14CystC were described previously in Examples1-7 above. Mouse NMuMG MECs and rat NRK kidney fibroblasts were infectedovernight with control (i.e., pMSCV-IRES-GFP), CystC, or Δ14CystCretroviral supernatants produced by EcoPac2 retroviral packaging cells(Clontech) as described above. Cells expressing GFP were isolated andcollected 48 h later on a MoFlo cell sorter (Cytomation), andsubsequently were expanded to yield stable polyclonal populations ofcontrol-, CystC, or Δ14CystC-expressing cells. The expression andsecretion of recombinant CystC proteins by infected NMuMG and NRK cellswas monitored by immunoblotting conditioned-media with anti-CystCantibodies as described above.

Immunofluorescence Studies

The ability of TGF-β to alter actin cytoskeletal architecture andE-cadherin expression was monitored essentially as described (Piek etal., 1999). Briefly, control-, CystC-, or Δ14CystC-expressing NMuMGcells were allowed to adhere overnight to glass coverslips in 24-wellplates (50,000 cells/well). The cells were stimulated the following daywith TGF-β1 (5 ng/ml) for 0-36 hours at 37° C. In some experiments,control NMuMG cells were stimulated with TGF-β1 in the absence orpresence of 10 μg/ml of recombinant GST, GST-CystC, or GST-Δ14CystC.Upon completion of agonist stimulation, the cells were washed inice-cold PBS and immediately fixed in 3.7% formaldehyde. After extensivewashing in PBS, the cells were blocked in PBS supplemented with 1.5%FBS, followed by incubation with rhodamine-phalloidin (0.25 μM).Alternatively, the cells were blocked in goat γ-globulin (200 μg/ml;Jackson Immunoresearch) prior to visualizing E-cadherin by sequentialincubations with monoclonal anti-E-cadherin (1:50 dilution; BDBioscience), followed by biotinylated goat anti-mouse antibody (5 μg/ml;Jackson Immunoresearch), and finally by Alexa-streptavidin (1.2 μg/ml;Molecular Probes). Images were captured on a Nikon Diaphot microscope.

The ability of TGF-β to alter E-cadherin expression also was monitoredby immunoprecipitating E-cadherin from Buffer H/1% TritonX-100-solubilized (Schiemann et al., 2003) NMuMG whole cell extracts,followed by immunoblotting with anti-E-cadherin antibodies (1:1000dilution).

Soft Agar Assay

The growth of NRK cells in soft agar was performed according to theprocedures described in (Moustakas et al., 1999). Briefly, duplicatecultures of control-, CystC-, or Δ14CystC-expressing NRK cells (10,000cells/plate) were grown in 0.3% agar on a cushion of 0.6% agar in 35-mmplates. NRK cell growth in the absence or presence of TGF-β1 (5 ng/ml)was allowed to proceed for 7 d, whereupon the number of colonies formedwas quantified under a light microscope.

Example 8

The following example demonstrates that CystC prevents TGF-β stimulationof EMT in NMuMG cells.

The importance of EMT in promoting cancer progression and tumormetastasis is becoming increasingly apparent (Thiery, 2002; Grunert,2003). Although the formation and growth of early stage tumors isnormally suppressed by TGF-β, cancer progression typically enables TGF-βto stimulate the growth and metastasis of late stage tumors, in partthrough its induction of EMT (Grunert, 2003). The inventor describes inExamples 1-7 above the cysteine protease inhibitor CystC as a novelTβR-II antagonist. Indeed, the physical association of CystC with TβR-IInot only prevented TGF-β binding and, consequently, TGF-β signaling innormal and cancer cells, but also inhibited their invasion throughsynthetic basement membranes. Collectively, these findings led theinventor to hypothesize CystC as a novel chemopreventive agent capableof antagonizing TGF-β oncogenicity in late stage tumors, particularlythe ability of TGF-β to induce EMT.

To test this hypothesis, the inventor first examined whether CystC and aCystC mutant impaired in its ability to inhibit cathepsin proteaseactivity (i.e., Δ14CystC) could antagonize TGF-β stimulation of EMT inMECs. In contrast to developing tissues, inappropriate induction of EMTby TGF-β in adult tissues enhances tumorigenesis. TGF-β stimulation ofEMT is studied routinely in murine NMuMG MECs, which readily undergo EMTwhen treated with TGF-β (Piek et al., 1999; Miettinen et al., 1994;Gotzmann et al., 2004). Briefly, NMuMG cells were stimulated with TGF-β1(5 ng/ml) for 0-24 hours in the presence of 10 μg/ml of either GST,GST-CystC, or GST-Δ14CystC. Afterward, altered actin cytoskeletalarchitecture was visualized by direct rhodamine-phalloidinimmunofluorescence. In a second experiment, NMuMG cells were stimulatedwith TGF-β1 (5 ng/ml) for 36 hours in the presence of GST fusionproteins as above. E-cadherin expression was monitored by indirectimmunofluorescence with anti-E-cadherin antibodies. The results (datanot shown) demonstrated that unstimulated NMuMG cells exhibited typicalepithelial cuboidal morphology characterized by strong cortical anddiffuse cytoplasmic actin staining In response to TGF-β, NMuMG cellstransition into fibroblasts and exhibited distinct actin stress fibersemanating from focal adhesions.

In another experiment, NMuMG cells were infected with ecotropicretrovirus encoding either GFP (i.e., control), CystC, or Δ14CystC, andsubsequently were isolated by FACS-sorting for GFP expression to yieldstable polyclonal populations of control, CystC, and Δ14CystC-expressingcells. The expression and secretion of recombinant CystC proteins byinfected NMuMG and NRK cells was monitored by immunoblottingconditioned-media with anti-CystC antibodies. The same cells wereincubated in the absence or presence of TGF-β1 (5 ng/ml) for 8 hours,whereupon alterations in actin cytoskeletal architecture was visualizedby direct rhodamine-phalloidin immunofluorescence. Next, the control,CystC-, and Δ14CystC-expressing NMuMG cells were incubated in theabsence or presence of TGF-β1 (5 ng/ml) for 36 hours, whereuponE-cadherin expression was monitored by immunoblotting withanti-E-Cadherin antibodies. Finally, control, CystC-, andΔ14CystC-expressing NMuMG cells were allowed to invade through Matrigelmatrices in the absence or presence of TGF-β1 (5 ng/ml) for 48 hours.

Recombinant CystC or Δ14CystC treatment of or their overexpression inNMuMG cells prevented actin cytoskeletal reorganization stimulated byTGF-β, as well as antagonized TGF-β-mediated downregulation ofE-cadherin (FIG. 5A; values depict downregulation of E-cadherinexpression induced by TGF-β relative to matched untreated NMuMG cells;this representative experiment was repeated twice with similar results).In addition, NMuMG cells undergoing EMT exhibit elevated invasionthrough synthetic basement membranes (FIG. 5B). Similar to their actionson 3T3-L1 cells, CystC and Δ14CystC expression also inhibited anddelineated cathepsin- and TGF-β-dependent invasion in NMuMG cells (FIG.5B). Values are the mean (±SE) of three independent experimentspresented as the percent invasion relative to GFP-expressing NMuMGcells. TGF-β1 significantly enhanced NMuMG cell invasion (*, p<0.05;Student's t-Test), a response that was inhibited significantly by CystCand Δ14CystC expression (#, p<0.05; Student's t-Test). CystC expressionalso significantly inhibited tonic NMuMG cell invasion (*, p<0.05;Student's t-Test).

Collectively, these findings show that CystC and Δ14CystC do indeedeffectively inhibit mammary cell EMT and invasion stimulated by TGF-β.Moreover, these findings suggest that future therapies employing CystCor Δ14CystC will prove effective in alleviating late stage tumorprogression stimulated by TGF-β.

Example 9

The following example shows that CystC prevents TGF-β stimulation ofmorphological transformation in NRK cells.

The loss of cell polarity and the ability of cancer cells to growautonomously in an anchorage-independent manner is a hallmark of cancer(Thiery, 2002; Grunert et al., 2003). TGF-β originally was described asa secreted factor that stimulates morphological transformation in ratNRK-49 kidney cells, leading to their acquisition ofanchorage-independent growth in soft agar (Roberts et al., 1981; Assoianet al., 1983). Thus, in addition to promoting EMT, TGF-β also enhancescancer progression by stimulating morphological transformation andanchorage-independent cell growth. Because CystC and Δ14CystC botheliminated EMT stimulated by TGF-β, the present inventor hypothesizedthat these TβR-II antagonists would similarly inhibit NRK cellmorphological transformation stimulated by TGF-β.

This hypothesis was tested by infecting NRK cells with bicistronicretrovirus encoding either CystC or Δ14CystC to determine their effectson NRK anchorage-independent growth stimulated by TGF-β. Briefly, ratNRK kidney fibroblasts were infected with control (i.e.,pMSCV-IRES-GFP), CystC, or Δ14CystC retroviral supernatants, and theresulting infected cells were isolated by GFP fluorescence on a MoFlocell sorter 48 hours later. Control, CystC-, and Δ14CystC-expressing NRKcells were cultured in soft-agar in the absence or presence of TGF-β1 (5ng/ml) for 7 d, whereupon NRK colony formation was quantified by lightmicroscopy. In addition, control, CystC-, and Δ14CystC-expressing NRKcells were allowed to invade through Matrigel matrices in the absence orpresence of TGF-β1 (5 ng/ml) for 48 hours.

As expected, TGF-β treatment enabled NRK cells to grow in ananchorage-independent manner when cultured in soft agar (FIG. 6A).Values are the mean (±SE) colony formation per microscope field observedin five independent experiments. Similar to their inhibitory activitiesin NMuMG cells, retroviral-mediated expression of CystC or Δ14CystC inNRK cells completely prevented morphological transformation stimulatedby TGF-β (FIG. 6A), as well as TGF-β stimulation of NRK cell invasionthrough synthetic basement membranes (FIG. 6B). Values are the mean(±SE) of three independent experiments presented as the percent invasionrelative to GFP-expressing NRK cells. TGF-β1 significantly enhanced NRKcell invasion (*, p<0.05; Student's t-Test). This TGF-β response wasinhibited significantly by CystC and Δ14CystC expression (#, p<0.05;Student's t-Test), while tonic NRK cell invasion was only inhibitedsignificantly by CystC expression (*, p<0.05; Student's t-Test).

Thus, in addition to preventing TGF-β stimulation of EMT, CystC andΔ14CystC also effectively inhibit morphological transformation andanchorage-independent growth stimulated by TGF-β.

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While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims:

1. An isolated Cystatin C homologue, wherein the amino acid sequence ofthe homologue differs from SEQ ID NO:2 by a deletion of amino acidresidues from about position 80 to about position 93 with reference toSEQ ID NO:2, wherein the Cystatin C homologue inhibits TGF-β biologicalactivity.
 2. An isolated protein comprising a fragment of SEQ ID NO:2that inhibits TGF-β biological activity, wherein the fragment differsfrom SEQ ID NO:2 by a deletion of at least 30 amino acids from theN-terminus of SEQ ID NO:2 and comprises at least the last 45 amino acidsof SEQ ID NO:2.
 3. The isolated protein of claim 2, wherein the proteininhibits the binding of TGF-β to its receptor.
 4. The isolated proteinof claim 3, wherein the receptor is TβRII.
 5. The isolated protein ofclaim 2, wherein the fragment comprises at least about 100 amino acidsof the C-terminal portion of SEQ ID NO:2.
 6. The isolated protein ofclaim 2, wherein the fragment comprises at least about 75 amino acids ofthe C-terminal portion of SEQ ID NO:2.
 7. The isolated protein of claim2, wherein the fragment comprises at least about 55 amino acids of theC-terminal portion of SEQ ID NO:2.
 8. The isolated homologue of claim 2,wherein the fragment differs from SEQ ID NO:2 by a deletion of at leastabout 40 amino acids from the N-terminus of SEQ ID NO:2.
 9. The isolatedhomologue of claim 2, wherein the fragment differs from SEQ ID NO:2 by adeletion of at least about 60 amino acids from the N-terminus of thewild-type protein.
 10. A therapeutic composition comprising the isolatedCystatin C homologue of claim 2, and a pharmaceutically acceptablecarrier.